Bispecific binding proteins and uses thereof

ABSTRACT

The disclosure generally provides proteins that bind two epitopes (e.g., a first and a second epitope) and that are bivalent for binding to each of the first and second epitopes. The disclosure also provides for specific binding proteins, including antibodies, which bind to a target protein. The disclosure also provides compositions comprising such proteins, nucleic acid molecules encoding such proteins and methods of making such proteins. The disclosure provides methods of inducing an immune response in a subject as well as methods for treating or preventing cancer in a subject by administering the proteins, nucleic acid molecules and/or compositions to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/332,788 filed May 6, 2016, the disclosure of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledIOBS_100_ST25.txt created May 2, 2017, which is 244 kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

Cancer continues to be a major global health burden. Despite progress inthe treatment of cancer, there continues to be an unmet medical need formore effective and less toxic therapies, especially for those patientswith advanced disease or cancers that are resistant to existingtherapeutics.

The role of the immune system, in particular T cell-mediatedcytotoxicity, in tumor control is well recognized. There is mountingevidence that T cells control tumor growth and survival in cancerpatients, both in early and late stages of the disease. However,tumor-specific T-cell responses are difficult to mount and sustain incancer patients. The continuing advancement and successes of cancerimmunotherapies, which stimulate or enhance innate immune responsesagainst cancer, make such therapeutics an attractive treatment optionwhen compared to therapies that utilize non-specific chemotherapeuticsand/or radiation.

A number of molecular targets have been identified for their potentialutility as immuno-oncology (IO) therapeutics against cancer. Somemolecular targets that are being investigated for their therapeuticpotential in the area of immuno-oncology therapy include cytotoxic Tlymphocyte antigen-4 (CTLA-4 or CD152), programmed death ligand 1 (PD-L1or B7-H1 or CD274), Programmed Death-1 (PD-1), OX40 (CD134 or TNFRSF4)and T-cell inhibitory receptor T-cell immunoglobulin and mucin-domaincontaining-3 (TIM3). While some of these targets have been successfullyexploited therapeutically (e.g., PD-1 and CTLA-4), many patients havebeen unresponsive to the therapeutics that have been developed. And,while a therapeutic regimen that includes higher doses and/or acombination of immunotherapies may be considered, such therapies may beassociated with increased risk of side effects, which tend to increasewith higher doses and cumulative exposure, and appear to be additivewhen used with combination immunotherapies. Some common side effectsinclude hypophysitis, thyroiditis, adrenal insufficiency, enterocolitis,dermatitis, pneumonitis, hepatitis, pancreatitis, motor and sensoryneuropathies, and arthritis. Furthermore, as immunotherapeutics aretypically associated with high costs, a therapy that includes acombination of immunotherapeutics can be cost-prohibitive to patients.

As such, there remains a need to continue to identify candidate targetsfor IO therapeutics, develop new therapeutics to the existing targets,and to develop therapeutic strategies that avoid disadvantages ofimmunotherapies that are currently in use, including the lack of patientresponse and the increased risk of side effects involved withcombination treatment. IO therapeutics (e.g., binding proteins) that arebispecific for a combination of target molecules, particularly thosethat exhibit greater binding affinity for the target molecules whencompared to the binding affinity for a combination of individualmonospecific binding proteins, represent a class of particularlydesirable molecules for therapeutic potential.

SUMMARY OF THE INVENTION

The invention provides bispecific molecules or proteins that bind twoepitopes (e.g., a first and a second epitope) and that are bivalent forbinding to each of the first and second epitopes. The invention alsoprovides methods of inducing an immune response in a subject as well asmethods for treating or preventing cancer in a subject (e.g., a humansubject) by administering the proteins, nucleic acid molecules and/orcompositions to the subject.

In one aspect, the invention provides a protein, containing: a firstbinding domain (BD1) that binds to a first epitope, a second bindingdomain (BD2) that binds to a second epitope, and an Fc region havingC_(H)2 and C_(H)3 domain; where the Fc region includes BD2 at a solventexposed loop in the C_(H)2 domain, the C_(H)3 domain, or at theinterface of the C_(H)2 and C_(H)3 domains; and where the protein isbivalent for binding to each of the first and second epitopes.

In another aspect, the invention provides a composition containing aprotein or antibody according to any aspect herein and apharmaceutically acceptable carrier.

In another aspect, the invention provides a method of treating orpreventing cancer in a subject, the method involving administering theprotein or antibody according to any aspect herein to the subject (e.g.,a human subject). In various embodiments, the cancer is one or more ofovarian cancer, breast cancer, colorectal cancer, prostate cancer,cervical cancer, uterine cancer, testicular cancer, bladder cancer, headand neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, andlung cancer.

In another aspect, the invention provides a method of inducing an immuneresponse in a subject, the method involving administering the protein orantibody according to any aspect herein to the subject (e.g., a humansubject).

In another aspect, the invention provides a nucleic acid molecule havinga nucleotide sequence encoding a protein or an antibody according to anyaspect herein.

In another aspect, the invention provides a vector containing a nucleicacid molecule according to any aspect herein.

In another aspect, the invention provides a host cell containing avector according to any aspect herein.

In one aspect, the invention provides a bispecific binding protein thatbinds to PD-1 and CTLA-4 having a first peptide having the amino acidsequence of SEQ ID NO:1, and a second peptide having the amino acidsequence of SEQ ID NO:2.

In another aspect, the invention provides a bispecific binding proteinthat binds to PD-1 and CTLA-4 having a first peptide having the aminoacid sequence of SEQ ID NO:3, and a second peptide having the amino acidsequence of SEQ ID NO:4.

In another aspect, the invention provides a bispecific binding proteinthat binds to PD-1 and CTLA-4 having, a first peptide having the aminoacid sequence of SEQ ID NO:5, and a second peptide having the amino acidsequence of SEQ ID NO:6.

In one aspect, the invention provides a bispecific binding protein thatbinds to PD-1 and CTLA-4 having a first heavy chain having the aminoacid sequence of SEQ ID NO: 9, a first light chain having the amino acidsequence of SEQ ID NO: 7, a second heavy chain having the amino acidsequence of SEQ ID NO: 12, and a second light chain having the aminoacid sequence of SEQ ID NO: 4.

In one aspect, the invention provides a bispecific binding protein thatbinds to PD-L1 and CTLA-4 having a first peptide having the amino acidsequence of SEQ ID NO:14 and a second peptide having the amino acidsequence of SEQ ID NO:15.

In another aspect, the invention provides a bispecific binding proteinthat binds to PD-L1 and CTLA-4 having a first peptide having the aminoacid sequence of SEQ ID NO:16, and a second peptide having the aminoacid sequence of SEQ ID NO:17.

In another aspect, the invention provides a bispecific binding proteinthat binds to PD-L1 and CTLA-4 having a first peptide having the aminoacid sequence of SEQ ID NO:18, and a second peptide having the aminoacid sequence of SEQ ID NO:19.

In one aspect, the invention provides a bispecific binding protein thatbinds to PD-1 and TIM3 having a first peptide having the amino acidsequence of SEQ ID NO:22, and a second peptide having the amino acidsequence of SEQ ID NO:23.

In another aspect, the invention provides a bispecific binding proteinthat binds to PD-1 and TIM3 having a first peptide having the amino acidsequence of SEQ ID NO:24 or SEQ ID NO:91, and a second peptide havingthe amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 92.

In one aspect, the invention provides a bispecific binding protein thatbinds to PD-1 and TIM3 having a first heavy chain having the amino acidsequence of SEQ ID NO:9, a first light chain having the amino acidsequence of SEQ ID NO: 7, a second heavy chain having the amino acidsequence of SEQ ID NO:27 or SEQ ID NO: 30, and a second light chainhaving the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28.

In one aspect, the invention provides a bispecific binding protein thatbinds to OX40 and PD-L1 having a first peptide having the amino acidsequence of SEQ ID NO:34, and a second peptide having the amino acidsequence of SEQ ID NO:32.

In another aspect, the invention provides a bispecific binding proteinthat binds to OX40 and PD-L1 having a first peptide having the aminoacid sequence of SEQ ID NO:35, and a second peptide having the aminoacid sequence of SEQ ID NO:32.

In another aspect, the invention provides a bispecific binding proteinthat binds to OX40 and PD-L1 having a first peptide having the aminoacid sequence of SEQ ID NO:36 or SEQ ID NO:94, and a second peptidehaving the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:93.

In one aspect, the invention provides an antibody or antigen bindingfragment thereof that binds to TIM3 having a heavy chain having CDR1,CDR2, and CDR3 and a light chain having CDR1, CDR2, and CDR3, whereinthe heavy chain CDR1 comprises SEQ ID NO:88, the heavy chain CDR2comprises SEQ ID NO:80, the heavy chain CDR3 comprises SEQ ID NO:81, andthe light chain CDR1 comprises SEQ ID NO:82, the light chain CDR2comprises SEQ ID NO:83, the light chain CDR3 comprises SEQ ID NO:84.

In other aspects, the invention provides a composition having abispecific binding protein and a pharmaceutically acceptable carrier; anucleic acid molecule having a nucleotide sequence encoding a bispecificbinding protein; methods of treating or preventing cancer in a subject,by administering a bispecific binding protein; and methods of enhancingan immune response in a subject, by administering a bispecific bindingprotein.

In various embodiments of any aspect delineated herein, the Fc regioncomprises BD2 at a solvent exposed loop in the amino acid sequence inthe C_(H)2 domain, the C_(H)3 domain, or at the interface of the C_(H)2and C_(H)3 domain.

In various embodiments of any aspect delineated herein, the solventexposed loop includes an amino acid sequence from the C_(H)2 domain. Inparticular embodiments, the solvent exposed loop includes the amino acidsequence ISRTP (SEQ ID NO: 39).

In various embodiments of any aspect delineated herein, the solventexposed loop includes an amino acid sequence from the C_(H)3 domain. Inparticular embodiments, the solvent exposed loop includes the amino acidsequence SNG.

In various embodiments of any aspect delineated herein, the solventexposed loop includes an amino acid sequence from the interface of theC_(H)2 domain and the C_(H)3 domain. In particular embodiments, theprotein of claim 7, where the solvent exposed loop comprises the aminoacid sequence AKGQP (SEQ ID NO: 40).

In various embodiments of any aspect delineated herein, BD2 is orincludes a single-chain variable fragment (scFv).

In various embodiments of any aspect delineated herein, BD1 is orincludes a binding domain that is one or more of an Fab domain, an scFv,a single domain antibody, and an antibody variable domain. In particularembodiments, BD1 includes a Fab domain.

In various embodiments of any aspect delineated herein, the Fab domainis connected to the Fc region via an antibody hinge region. In certainembodiments, the Fc region is or includes a domain that is one or moreof an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.In particular embodiments, the Fc region comprises a variant Fc region.In some embodiments, the Fc region is aglycosylated, deglycosylated,and/or is afucosylated or has reduced fucosylation.

In various embodiments of any aspect delineated herein, the proteinfurther includes a protein linker L1 between BD2 and the Fc region. Invarious embodiments of any aspect delineated herein, the protein furtherincludes a first protein linker, L1, and a second protein linker, L2,between BD2 and the Fc region. In various embodiments of any aspectdelineated herein, BD2 is associated with the Fc region via a proteinlinker L1. In various embodiments of any aspect delineated herein, BD2is associated with the Fc region via two protein linkers, L1 and L2. Incertain embodiments, L1 and L2 are independently selected from (G₄S)₂(SEQ ID NO:41), (G₄S)₃, (SEQ ID NO:42), and (G₄S)₄ (SEQ ID NO:43).

In various embodiments of any aspect delineated herein, the proteinincludes a chimeric heavy chain having the following polypeptidedomains, from N-terminus to C-terminus: V_(H)1-C_(H)1-C_(H)2(N-term)-BD2-C_(H)2 (C-term)-C_(H)3; and BD1 includes a Fab domain;where V_(H)1 includes a heavy chain variable domain of the Fab domainand C_(H)1 includes the heavy chain constant domain 1 of the Fab.

In various embodiments of any aspect delineated herein, the proteinincludes a chimeric heavy chain having the following polypeptidedomains, from N-terminus to C-terminus: V_(H)1-C_(H)1-C_(H)2-BD2-C_(H)3;and BD1 includes a Fab domain; where V_(H)1 comprises a heavy chainvariable domain of the Fab domain and C_(H)1 includes the heavy chainconstant domain 1 of the Fab.

In various embodiments of any aspect delineated herein, the proteinincludes a chimeric heavy chain having the following polypeptidedomains, from N-terminus to C-terminus:V_(H)1-C_(H)1-C_(H)2-C_(H)3(N-term)-BD2-C_(H)3(C-term); and BD1 includesa Fab domain; where V_(H)1 includes a heavy chain variable domain of theFab domain, and C_(H)1 includes the heavy chain constant domain 1 of theFab.

In various embodiments of any aspect delineated herein, BD2 is orincludes an scFv. In particular embodiments, the scFv includes, fromN-terminus to C-terminus: V_(H)2-polypeptide linker-V_(L)2 orV_(L)2-polypeptide linker-V_(H)2; where V_(H)2 includes the heavy chainvariable domain of the scFv and V_(L)2 includes the light chain variabledomain of the scFv.

In various embodiments of any aspect delineated herein, the proteinfurther includes a protein linker L1 between BD2 and the Fc region. Invarious embodiments of any aspect delineated herein, the protein furtherincludes a first protein linker, L1, and a second protein linker, L2,between BD2 and the Fc region.

In various embodiments of any aspect delineated herein, the BD2 isassociated via a linker (L1) to the C_(H)2 domain, the C_(H)2 domain, orthe interface of the C_(H)2 and C_(H)3 domains of the Fc region.

In various embodiments of any aspect delineated herein, the BD2 isassociated via two protein linkers, L1 and L2 to the C_(H)2 domain, theC_(H)3 domain, or the interface of the C_(H)2 and C_(H)3 domains of theFc region. In various embodiments, L1 and L2 are independently selectedfrom protein linkers having a length of 1-25 amino acids. In particularembodiments, L1 and L2 are independently selected from (G₄S)₂ (SEQ IDNO:41), (G₄S)₃, (SEQ ID NO:42), and (G₄S)₄ (SEQ ID NO:43).

In various embodiments of any aspect delineated herein, the first andsecond epitopes are different. In various embodiments of any aspectdelineated herein, the first and second epitopes are the same.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the disclosure, there are depicted inthe drawings certain aspects of the disclosure. However, the disclosureis not limited to the precise arrangements and instrumentalities of theaspects depicted in the drawings.

FIGS. 1A-1F depict a general schematic diagram of certain exemplaryproteins described herein. The CH2 and CH3 regions are illustrated inFIGS. 1A-1C using PyMOL and identify solvent exposed surface loopregions as spheres. FIG. 1A depicts the loop in the CH2 region; FIG. 1Bdepicts the loop in the CH2-CH3 interface; and FIG. 1C depicts the loopin the CH3 region. Exemplary constructs are illustrated in FIGS. 1D-1Fthat include representative BD1 and BD2 domains as Fab and scFv domains,respectively. FIG. 1D depicts BD1 attached at the hinge region and BD2attached at solvent exposed loops in the CH2 region. FIG. 1E depicts BD1attached at the hinge region and BD2 attached at solvent exposed loopsin the CH2-CH3 interface. FIG. 1F depicts BD1 attached at the hingeregion and BD2 attached at solvent exposed loops in the CH3 region.

FIGS. 2A-2C provide an expanded view of the solvent accessible loopsequences in CH2, at the CH2-CH3 interface, and in CH3 as describedherein. Examples of constructs incorporating a BD2 (scFv) are includedin each of FIGS. 2A-2C. FIG. 2A illustrates the representative loopsequence ISRTP (SEQ ID NO:39) identified in the CH2 loop upstream of theCH2-CH3 interface. FIG. 2B illustrates the representative loop sequenceAKGQP (SEQ ID NO:40) in the CH2-CH3 interface. FIG. 2C illustrates therepresentative loop sequence SNG in the CH3 region downstream of theCH2-CH3 interface.

FIG. 3 demonstrates the concurrent binding of the BiS2, BiS3, and BiS5constructs that target PD-1 and CTLA-4. Trace A9 shows BiS2 PD-1/CTLA-4,trace B9 shows Bis3 PD-1/CTLA-4, and trace C9 shows Bis5 PD-1/CTLA-4.

FIG. 4 shows a schematic of the proposed mechanism for the PD-1/CTLA-4blockade.

FIG. 5 shows the results of an octet binding assay which demonstratesthe concurrent binding of a DuetMab construct that targets PD-1 andCTLA-4.

FIGS. 6A-D shows that PD-1/CTLA-4 bispecific binding proteins inhibitthe PD-1 and CTLA-4 pathways in reporter gene assays. FIG. 6A depictsT-cell activation via PD-1 blockade. FIG. 6B depicts T-cell activationvia CTLA-4 blockade. FIG. 6C shows the results of the PD-1 reporterassay. FIG. 6D shows the results of the CTLA-4 reporter assay.

FIG. 7 shows the results of a SEB assay showing that PD-1/CTLA-4 DuetMaband BiS5Ab have equivalent activity in the Staphylococcal enterotoxin B(SEB) assay.

FIGS. 8A-B show the activity of PD-1/CTLA-4 DuetMab in SEB assayscompared to isotype and parental mAb controls.

FIGS. 9A-B show activity of PD-1/CTLA-4 BiS5Ab compared to PD-1/CTLA-4DuetMab in SEB assays.

FIGS. 10A-C show that PD-1/CTLA-4 DuetMab and BiS5Ab have equivalentactivity in the mixed lymphocyte reaction (MLR) assay. FIG. 10A is aschematic of the assay (n=4 donors; 2 independent experiments).

FIGS. 11A-D show the activity of PD-1/CTLA-4 DuetMab in MLR assayscompared to isotype controls (n=2 donors; 1 experiment).

FIG. 12A-D show the activity of PD-1/CTLA-4 DuetMab in MLR assayscompared to parental mAb controls (n=2 donors; 1 experiment).

FIGS. 13A-D shows the activity of PD-1/CTLA-4 DuetMab in MLR assayscompared to competitor antibodies (n=2 donors; 1 experiment).

FIG. 14 shows the study design for a single dosepharmacokinetic/pharmacodynamic (PK/PD) study in cynomolgus monkeys.

FIGS. 15A-B show that PD-1/CTLA-4 DuetMab showed clear pharmacodynamics(PD) in cynomolgus monkeys.

FIG. 16A-B shows T cell dependent antibody response (TDAR) in cynomolgusmonkeys dosed with PD-1/CTLA-4 DuetMab (MEDI5752) and PD-1/CTLA-4 BiS5Ab(MEDI8500).

FIG. 17 shows a model system to study PD-1/CTLA-4 bispecific moleculesin which stable CHO cells express diverse levels of human PD-1 and/orCTLA-4.

FIGS. 18A-C show that PD-1/CTLA-4 DuetMab concurrently binds PD-1 andCTLA-4 on the surface of the same cell.

FIGS. 19A-C show an experiment to determine whether co-operative bindingdifferentiates over a combination of anti-PD-1 and anti-CTLA-4antibodies in the saturation of CTLA-4 on cells expressing excess PD-1.

FIGS. 20A-D show that PD-1 and CTLA-4 parental monoclonal antibodiesbind and occupy their target receptor without a measurable effect on theuntargeted receptor.

FIGS. 21A-D show that PD-1/CTLA-4 DuetMab saturates CTLA-4 on CHO cellsexpressing excess PD-1 at ˜250-fold lower concentrations compared to acombination of monoclonal antibodies.

FIGS. 22A-F show that PD-1/CTLA-4 DuetMab saturates CTLA-4 on CHO cellsexpressing excess PD-1 at ˜500-fold lower concentrations compared tocells expressing only CTLA-4.

FIGS. 23A-B shows PD-1/CTLA-4 DuetMab preferentially binds in cis toPD-1 and CTLA-4 on the surface of same cell. Treme in FIG. 23B is aCTLA-4 mAb.

FIGS. 24A-D show binding and internalization of PD-1/CTLA-4 DuetMab andparental monoclonal antibodies to cultured T cells. PD-1/CTLA-4 DuetMabhas internalization properties of tremelimumab.

FIG. 25A shows a schematic of the internalization assay. FIG. 25B showsthat PD-1/CTLA-4 DuetMab takes on internalization properties oftremelimumab in stable CHO cells expressing 10-fold excess PD-1.

FIG. 26 demonstrates the concurrent binding of the BiS2, BiS3, and BiS5constructs that target PD-L1 and CTLA-4. Trace A11 shows Bis2PD-L1CTLA-4, trace B11 shows Bis3 PD-L1/CTLA-4, and trace C11 shows Bis5PD-L1/CTLA-4.

FIG. 27A demonstrates the concurrent binding of the BiS3 construct thattargets PD-1 and TIM3 (clone 62, wild type). FIG. 27B demonstrates theconcurrent binding of a DuetMab construct that targets PD-1 and TIM3(clone 62, wild type).

FIGS. 28A-28C provide a summary of the cell killing activity ofmonospecific TIM3 and bispecific PD-1_TIM3PD-1 bispecific constructs ina cell killing assay. FIG. 28A shows brightfield images of coculturedwells at 18 hr. Combination of anti-TIM-3+anti-PD1 or the TIM-3/PD-1bispecific formats enhance tumor cell death and increase T cellactivation as assessed by reduction of adherent cells and enhancedblasting (clumping) of T cells. FIG. 28B shows assessment of viabilitydye uptake by tumor cells after 18 hr co-culture with melanoma specificCD8+ T cells. FIG. 28C shows IFNγ secretion after 18 hr co-culture. Thebispecific constructs generally demonstrate better killing activity,with the DuetMab format exhibiting the most robust killing activity.

FIG. 29 demonstrates the concurrent binding of a PD-1/TIM3 DuetMabconstruct having a TIM3 arm sequence that is an affinity mature variantof clone 62.

FIG. 30 shows binding of PD-1/TIM3 bispecific antibodies, includingBiS3, BiS5, and DuetMab, to CHO cells overexpressing human TIM3 or humanPD1. PD-1 and TIM3 expression data are shown in the inset.

FIG. 31 shows TIM3 and PD-1 expression data on activated T cell clone(DMF4).

FIGS. 32A-C depict the results from a CMV antigen recall assay showingthat PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, and DuetMab,demonstrate enhanced activity compared to isotype treatment (3 donors(1-2 replicates per treatment/per donor), 1 experiment).

FIGS. 33A-D show that PD-1/TIM3 bispecific antibodies, including BiS3,BiS5, and DuetMab, enhanced interferon (IFNγ) at concentrations above 8nM in the mixed lymphocyte reaction (MLR) assay (2-4 replicate wells pertreatment/1 donor pair/1 of 2 independent experiments).

FIGS. 34A-C show the results of a PD-1 reporter assay (dual cell system)using PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, andDuetMab, All bispecific formats demonstrate similar activity as parentalLO115 IgG1 (compilation of 3-5 independent experiments/3 biologicalreplicates per treatment).

FIG. 35 shows the results of an octet assay using BiS2 and BiS3OX40/PD-L1 bispecific molecules.

FIGS. 36A-B show the results of an SEB assay using BiS2 and BiS3OX40/PD-L1 bispecific molecules.

FIG. 37 shows the results of a PD-L1 reporter assay using BiS2 and BiS3OX40/PD-L1 bispecific molecules.

FIG. 38 shows the results of a CMV Ag recall assay using BiS2 and BiS3OX40/PD-L1 bispecific molecules.

FIG. 39 shows the results of an octet binding assay which demonstratesthe concurrent binding of the OX40(SLR)/PD-L1 BiS5 construct thattargets PD-L1 and OX40.

FIGS. 40A-F shows binding of the bispecific constructs that target PD-L1and OX40 to CHO cells expressing human or cynomolgus OX40 andPD-L1/B7H1.

FIGS. 41A-B shows binding of the bispecific constructs that target PD-L1and OX40 to Jurkat OX40 reporter cells, NCI H358, CHOK1 B7H1(PD-L1)/OKT3cells, and HEK CD32a cells, measured by flow cytometry (HyperCyt).

FIGS. 42A-B show the results of a PD-L1 reporter assay using OX40/PD-L1bispecific molecules.

FIGS. 43A-B shows the results of an OX40 reporter assay in HEK CD32acells using OX40/PD-L1 bispecific molecules.

FIGS. 44A-B shows the results of an OX40 reporter assay in CHOK PD-L1overexpressing cells using OX40/PD-L1 bispecific molecules.

FIGS. 45A-B shows PD-L1 mediated OX40 agonism with tumor cells usingOX40/PD-L1 bispecific molecules.

FIGS. 46A-D shows results indicating no agonism was detected with NCIH358 PD-L1 KO cells using OX40/PD-L1 bispecific molecules in controlsfor the OX40/tumor cell assay.

FIGS. 47A-D shows the results of an SEB assay using OX40/PD-L1bispecific molecules.

FIG. 48A shows a schematic of the Treg suppression assay experiment totest the OX40/PD-L1 bispecific molecules. FIGS. 48B-C show the Tregsuppression based on binding of the bispecific molecule.

FIG. 49 depicts shows the results of the Treg suppression assay usingthe OX40/PD-L1 bispecific molecules.

FIG. 50 depicts shows the results of the Treg suppression assay usingthe OX40/PD-L1 bispecific molecules.

FIGS. 51A-B shows the design of a mixed leukocyte reaction (MLR) assayexperiment to test OX40/PD-L1 bispecific molecules.

FIGS. 52A-E shows the results of the MLR assay using the OX40/PD-L1bispecific molecules.

FIGS. 53A-B shows that BiS2 and BiS5 OX40/PD-L1 bispecific moleculesmediate antibody-dependent cell-mediated cytotoxicity (ADCC) of naturalkiller (NK) cells against PD-L1 or OX40 expressing CHO cells.

FIG. 54 shows that BiS2 and BiS5 OX40/PD-L1 bispecific molecules mediateantibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells againstPD-L1 and OX40 expressing CHO cells.

FIG. 55 shows that BiS2 and BiS5 OX40/PD-L1 bispecific moleculesincreased CD107a mobilization of NK cells against PD-L1 and OX40expressing CHO cells in antibody-dependent cell-mediated cytotoxicity(ADCC).

FIG. 56 shows that BiS2 and BiS5 OX40/PD-L1 bispecific molecules mediateantibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells againstactivated allogeneic T cells.

FIGS. 57A-B shows that BiS5 OX40/PD-L1 increased CD107a mobilization ofNK cells from two different donors against activated allogeneic T cellsin antibody-dependent cell-mediated cytotoxicity (ADCC).

FIG. 58 shows a study design to compare PK/PD of OX40/PD-L1 bispecificmolecules.

FIG. 59 shows a comparison of serum concentration time profiles forPD-L1/OX40 bispecific molecules in cynomolgus monkeys.

FIG. 60 shows depletion of soluble PD-L1 in serum by PD-L1/OX40bispecific molecules.

FIGS. 61A-F provides a summary of pharmacodynamic data for PD-L1/OX40bispecific molecules. Baseline defined as mean of Day −5 and Day 0pre-dose.

FIG. 62 depicts a schematic of a PD-1/OX40 BiS2 IgG4P monoclonalantibody (mAb).

FIG. 63 depicts a potential mechanism of action of PD-1/OX40 BiS2 mAb.

FIG. 64 depicts the concurrent binding activity for two different lotsof the PD-1(LO115)/OX40 BiS2 mAb to PD1-His and human OX40-Fc.

FIG. 65A depicts a schematic of an OX40 reporter assay. FIG. 65B showsthe results of the OX40 reporter assay using PD1 LO115 mAb, OX40 mAb, acontrol mAb and PD1(LO115)/OX40 BiS2 mAb.

FIG. 66A depicts a schematic of a PD-1/PD-L1 reporter assay. FIG. 66Bshows results of the PD1/PD-L1 reporter assay using PD1 LO115 mAb, OX40mAb, a control mAb and PD1(LO115)/OX40 BiS2 mAb.

FIG. 67 shows the results of an SEB assay using the BiS2 variant ofPD-1(LO115)/OX40 bispecific molecule and controls.

FIG. 68 shows the results of a CMV antigen recall assay using the BiS2variant of PD-1(LO115)/OX40 bispecific molecule and controls.

FIG. 69 shows the results of a CMV antigen recall assay using the BiS2and BiS3 variants of PD-1(AMP514)/OX40 bispecific molecules andcontrols.

FIG. 70 shows Serum concentration-time profiles of PD1(LO115)/OX40 BiS2mAb after single IV dose in cynomolgus monkeys. Data represents themean±standard deviation of 3 males/group. The LLOQ (5 ng/mL is shown bythe dotted line. PKC=pharmacokinetic concentration; LLOQ=lower limit ofquantitation.

FIG. 71 shows percent Ki67 positive CD4+ and CD8+ memory T cells aftersingle IV administration of PD-1(LO115)/OX40 BiS2 mAb in cynomolgusmonkeys. Data represent the mean±standard deviation of 3 males/group.Left panel A represents CD4+ memory T cells and right panel shows CD8+memory T cells. IV=intravenous.

FIG. 72 shows a representative standard curve for quantitation ofPD-1/OX40 in cynomolgus monkey serum.

FIGS. 73A-73E provide illustrative DSC thermograms of the bispecificbinding protein disclosed herein (“BiS5”) relative to a different BiSformat (“BiS4”) at different pH values. FIG. 73A illustrates the effectof pH on thermal stability of BiS4. FIG. 73B illustrates the effect ofpH on thermal stability of BiS5. FIG. 73C depicts a representativecurve-fitted DSC thermogram for BiS4 with T_(onset), T_(m)1, T_(m)2, andT_(m)3. FIG. 73D depicts a representative curve-fitted DSC thermogramsfor BiS5 with T_(onset), T_(m)1, T_(m)2, and T_(m)3. FIG. 73E depicts aplot representing the effect of pH on T_(onset), T_(m)1, T_(m)2, andT_(m)3 for BiS4 and BiS5 formats.

FIGS. 74A and 74B depict HP-SEC analysis of samples at pH 7.5 before andafter storage at 40° C. for 4 weeks. FIG. 74A provides an overlay plotof SEC chromatograms of BiS4 and BiS5 before and after thermal stress,the solid lines correspond to BiS4 and BiS5 samples at time zero (nostress) and dotted lines correspond to BiS4 and BiS5 samples incubatedat 40° C. for 4 week (stressed). FIG. 74B provides a bar chartrepresenting the effect of pH 7.5 on different species (monomer,fragments, and aggregates) of BiS4 and BiS5 as measured on day zero andafter 4 weeks at 40° C.

FIGS. 75A-75C provide kinetics plots showing the effect of pH 7.5 onaccelerated and short-term storage stability at 40° C. for BiS4(triangle trace) and BiS5 (circle trace). FIG. 75A shows the percentageof monomer remaining, as measured by HP-SEC over a period of 4 weeks.FIG. 75B shows the percentage of fragmentation, as measured by HP-SECover a period of 4 weeks. FIG. 75C shows the percentage of aggregation,as measured by HP-SEC over a period of 4 weeks. The data presented isfrom single vial analysis.

FIGS. 76A-76C depict pH rate profile plots for BiS4 (triangles) and BiS5(circles). FIG. 76A shows the effect of different pH conditions on therate of monomer loss. at 40° C. FIG. 76B shows the effect of differentpH conditions on the rate of fragmentation at 40° C. FIG. 76C shows theeffect of different pH conditions on the rate of aggregation at 40° C.

FIGS. 77A and 77B depict additional analysis of BiS4 and BiS5fragmentation. FIG. 77A shows that at pH 7.5 and 40° C. (at time=0),neither molecule exhibits appreciable fragmentation. FIG. 77B shows thatunder the same conditions as FIG. 77A, but after 2 weeks storage at 40°C., appreciable fragmentation is observed for BiS4 and minimalfragmentation for BiS5.

FIG. 78 depicts analysis of BiS4 and BiS5 fragmentation (left panel) andaggregation (right panel) as a function of pH. Both formats have reducedfragmentation and aggregation at lower (5.5) pH, while BiS5 has superiorperformance at both pH values for both fragmentation and aggregation.

FIG. 79 depicts DSC thermograms of several of the BiS5 bispecificbinding protein disclosed herein for constructs (left panel) A, B, C,and D, as well as (right panel) E, F, G, and H. Constructs A and Einclude the scFv at IS-RTP; B and F include the scFv at AK-GQP; C and Ginclude the scFv at S-NG; and D and H include the scFv at SN-G. The scFvfor constructs A, B, C, and D is 2F4 (IgG is LC10), while the scFv forE, F, G, and H is LC10 (IgG is 2F4). The various T_(M) values areassociated with the following domains, T_(M)1=CH2/scFv; T_(M)2=Fab;T_(M)3=CH3.

FIG. 80 depicts a representative data set for FcRn binding with thebispecific binding protein disclosed herein (constructs D and H). Thelocation of the scFv in the CH2-CH3 domain (i.e., within the ISTRP loop)can have an effect on FcRn binding activity.

FIG. 81 depicts a representative data set for FcγR binding with thebispecific binding protein disclosed herein (constructs E, G, and H withFcγRIIIa-158V). The inset reflects the same data as the main figure,renormalized at the FcγRIIIa-158V injection. All constructs were able tobind FcγRs with an observable difference in affinities based on locationof the scFv domains.

FIG. 82 shows that an intact ISRTP loop is important for FcRn binding ofthe illustrative bispecific binding constructs (e.g., A and E).Introduction of the N3 loop does not compensate for interruption of theISRTP loop (BiS5E+N3). An scFv introduced into an N3 loop inserted intoan IgG1 Fc renders the IgG unable to bind FcRn. Time in the x-axis ismeasured in seconds.

FIG. 83 depicts the general schematic structural format of each BiS1,BiS2, BiS3, BiS4, and BiS5 constructs. The denotations of “k1” and “k2”indicate the fragmentation patterns as used in the kinetic analysisdiscussed in Example 3.

FIG. 84 depicts the fragmentation rate of each BiS1, BiS2, BiS3, BiS4,and BiS5 as a function of pH.

FIG. 85 depicts the aggregation rate of each BiS1, BiS2, BiS3, BiS4, andBiS5 as a function of pH.

FIG. 86 depicts the monomer loss rate of each BiS1, BiS2, BiS3, BiS4,and BiS5 as a function of pH.

FIG. 87 depicts a representation of the fragmentation pattern and thecorrespondence to the peaks on HPSEC chromatograms for of each BiS1,BiS2, BiS3, BiS4, and BiS5.

FIG. 88 depicts representative analysis of the fragmentation patternunder reducing conditions.

FIG. 89 depicts the structural arrangement of BiS5 under reducing andnon-reducing conditions.

FIG. 90 depicts the SEB assay format.

DETAILED DESCRIPTION

Before continuing to describe the present disclosure in further detail,it is to be understood that this disclosure is not limited to specificcompositions or process steps, as such may vary. It must be noted that,as used in this specification and the appended claims, the singular form“a”, “an” and “the” include plural referents unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisinvention.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The numbering of amino acids in the variable domain, complementaritydetermining region (CDRs) and framework regions (FR), of an antibodyfollow, unless otherwise indicated, the Kabat definition as set forth inKabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insertion (residue 52a according to Kabat) after residue 52 of H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence. Maximal alignment of framework residues frequentlyrequires the insertion of “spacer” residues in the numbering system, tobe used for the Fv region. In addition, the identity of certainindividual residues at any given Kabat site number may vary fromantibody chain to antibody chain due to interspecies or allelicdivergence.

As used herein, the terms “antibody” and “antibodies”, also known asimmunoglobulins, encompass monoclonal antibodies (including full-lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodiesformed from at least two different epitope binding fragments (e.g.,multispecific antibodies, e.g., PCT publication WO2009018386, PCTApplication No. PCT/US2012/045229, incorporated herein by reference inits entirety), BiSAbs, human antibodies, humanized antibodies, camelisedantibodies, single-chain Fvs (scFv), single-chain antibodies, singledomain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments,antibody fragments that exhibit the desired biological activity (e.g.the antigen binding portion), disulfide-linked Fvs (dsFv), andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), intrabodies, and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain at least oneantigen-binding site. Antibodies also include peptide fusions withantibodies or portions thereof such as a protein fused to an Fc domain.Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM,IgD, IgA and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or allotype (e.g., Gm, e.g., G1m(f, z, a or x), G2m(n), G3m(g, b,or c), Am, Em, and Km(1, 2 or 3)). Antibodies may be derived from anymammal, including, but not limited to, humans, monkeys, pigs, horses,rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g.chickens).

CTLA-4

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed onactivated T cells and serves as a co-inhibitor to keep T-cell responsesin check following CD28-mediated T cell activation. CTLA-4 is believedto regulate the amplitude of the early activation of naïve and memory Tcells following TCR engagement and to be part of a central inhibitorypathway that affects both antitumor immunity and autoimmunity. CTLA-4 isexpressed exclusively on T cells, and the expression of its ligands CD80(B7.1) and CD86 (B7.2), is largely restricted to antigen-presentingcells, T cells, and other immune mediating cells. Antagonisticanti-CTLA-4 antibodies that block the CTLA-4 signaling pathway have beenreported to enhance T-cell activation. One such antibody, ipilimumab,was approved by the FDA in 2011 for the treatment of metastaticmelanoma. The use of anti-CTLA-4 antibodies to treat infections andtumors and up-modulate an adaptive immune response has been proposed(see, U.S. Pat. Nos. 6,682,736; 7,109,003; 7,132,281; 7,411,057;7,824,679; 8,143,379 7,807,797; 8,491,895; 8,883,984; and US PublicationNo. 20150104409, incorporated herein by reference in their entireties).

PD-L1

Programmed Death Ligand 1 (PD-L1) is also part of a complex system ofreceptors and ligands that are involved in controlling T-cellactivation. In normal tissue, PD-L1 is expressed on T cells, B cells,dendritic cells, macrophages, mesenchymal stem cells, bonemarrow-derived mast cells, as well as various non-hematopoietic cells.Its normal function is to regulate the balance between T-cell activationand tolerance through interaction with its two receptors: programmeddeath 1 (also known as PD-1 or CD279) and CD80 (also known as B7-1 orB7.1). PD-L1 is also expressed by tumors and acts at multiple sites tohelp tumors evade detection and elimination by the host immune system.PD-L1 is expressed in a broad range of cancers with a high frequency. Insome cancers, expression of PD-L1 has been associated with reducedsurvival and unfavorable prognosis. Antibodies that block theinteraction between PD-L1 and its receptors are able to relievePD-L1-dependent immunosuppressive effects and enhance the cytotoxicactivity of antitumor T cells in vitro. Durvalumab is a human monoclonalantibody directed against human PD-L1 that is capable of blocking thebinding of PD-L1 to both the PD-1 and CD80 receptors. The use ofanti-PD-L1 antibodies to treat infections and tumors and enhance anadaptive immune response has been proposed (see, U.S. Pat. Nos.8,779,108 and 9,493,565 incorporated herein by reference in theirentirety).

PD-1

Programmed Death-1 (“PD-1”) is an approximately 31 kD type I membraneprotein member of the extended CD28/CTLA-4 family of T cell regulators(see, Ishida, Y. et al. (1992) Induced Expression Of PD-1, A NovelMember Of The Immunoglobulin Gene Superfamily, Upon Programmed CellDeath,” EMBO J. 11:3887-3895.

PD-1 is expressed on activated T cells, B cells, and monocytes (Agata,Y. et al. (1996) “Expression of the PD-1 Antigen on the Surface ofStimulated Mouse T and B Lymphocytes,” Int. Immunol. 8(5):765-772;Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation and Anti-TumorImmunity,” Semin. Cancer Biol. 17(4):288-298). PD-1 is a receptorresponsible for down-regulation of the immune system followingactivation by binding of PDL-1 or PDL-2 (Martin-Orozco, N. et al. (2007)“Inhibitory Costimulation and Anti-Tumor Immunity,” Semin. Cancer Biol.17(4):288-298) and functions as a cell death inducer (Ishida, Y. et al.(1992) “Induced Expression of PD-1, A Novel Member of The ImmunoglobulinGene Superfamily, Upon Programmed Cell Death,” EMBO J. 11: 3887-3895;Subudhi, S. K. et al. (2005) “The Balance of Immune Responses:Costimulation Verse Coinhibition,” J. Molec. Med. 83: 193-202). Thisprocess is exploited in many tumours via the over-expression of PD-L1,leading to a suppressed immune response.

PD-1 is a well-validated target for immune mediated therapy in oncology,with positive results from clinical trials in the treatment of melanomaand non-small cell lung cancers (NSCLC), among others. Antagonisticinhibition of the PD-1/PD-L-1 interaction increases T-cell activation,enhancing recognition and elimination of tumour cells by the host immunesystem. The use of anti-PD-1 antibodies to treat infections and tumorsand enhance an adaptive immune response has been proposed (see, U.S.Pat. Nos. 7,521,051; 7,563,869; 7,595,048).

OX40

OX40 (CD134; TNFRSF4) is a tumor necrosis factor receptor foundprimarily on activated CD4⁺ and CD8⁺ T cells, regulatory T (Treg) cellsand natural killer (NK) cells (Croft et al., 2009, Immunol Rev.229:173-91). OX40 has one known endogenous ligand, OX40 ligand (OX40L;CD152; TNFSF4), which exists in a trimeric form and can cluster OX40,resulting in potent cell signaling events within T cells. Id. Signalingthrough OX40 on activated CD4⁺ and CD8⁺ T cells leads to enhancedcytokine production, granzyme and perforin release, and expansion ofeffector and memory T cell pools (Jensen et al., 2010, Semin Oncol.37:524-32). In addition, OX40 signaling on Treg cells inhibits expansionof Tregs, shuts down the induction of Tregs and blocks Treg-suppressivefunction (Voo et al., 2013, J Immunol. 191:3641-50; Vu et al., 2007,Blood. 110:2501-10).

Immunohistochemistry studies and early flow cytometry analyses showedthat OX40 is expressed on T cells infiltrating a broad range of humancancers (Baruah et al., 2011, Immunobiology 217:668-675; Curti et al,2013, Cancer Res. 73:7189-98; Ladanyi et al, 2004, Clin Cancer Res.10:521-30; Petty et al, 2002, Am J Surg. 183:512-8; Ramstad et al, 2000,Am J Surg. 179:400-6; Sarff et al, 2008, Am J Surg. 195:621-5;discussion 625; Vetto et al, 1997, Am J Surg. 174:258-65). While notwishing to be bound by theory, OX40 expression on tumor-infiltratinglymphocytes correlates with longer survival in several human cancers,suggesting that OX40 signals can play a role in establishing anantitumor immune response (Ladanyi et al., 2004, Clin Cancer Res.10:521-30; Petty et al., 2002, Am J Surg. 183:512-8).

In a variety of nonclinical mouse tumor models, agonists of OX40,including antibodies and OX40 ligand fusion proteins, have been usedsuccessfully with promising results (Kjaergaard et al., 2000, CancerRes. 60:5514-21; Ndhlovu et al., 2001, J Immunol. 167:2991-9; Weinberget al., 2000, J Immunol. 164:2160-9). Co-stimulating T cells throughOX40 promoted anti-tumor activity that in some cases was durable,providing long-lasting protection against subsequent tumor challenge(Weinberg et al., 2000, J Immunol. 164:2160-9). Treg-cell inhibition andco-stimulation of effector T cells were shown to be necessary for tumorgrowth inhibition of OX40 agonists (Piconese et al., 2008, J Exp Med.205:825-39). Many strategies and technologies have been explored toenhance the anti-tumor effect of OX40 agonist therapy throughcombinations with vaccines, chemotherapy, radiotherapy, andimmunotherapy (Jensen et al., 2010, Semin Oncol. 37:524-32; Melero etal., 2013, Clin Cancer Res. 19:997-1008). The use of anti-OX40antibodies to treat infections and tumors and up-modulate an adaptiveimmune response has been proposed (see, US Publication No. 20160137740,incorporated herein by reference in its entirety).

TIM3

The T-cell inhibitory receptor Tim-3 (T-cell immunoglobulin andmucin-domain containing-3) plays a role in regulating antitumor immunityas it is expressed on IFN-gamma producing CD4+ helper 1 (Th1) and CD8+ Tcytotoxic1 (Tc1) T cells. It was initially identified as a T-cellinhibitory receptor, acting as an immune checkpoint receptor thatfunctions specifically to limit the duration and magnitude of Th1 andTc1 T-cell responses. Further research has identified that the Tim-3pathway may cooperate with the PD-1 pathway to promote the developmentof a severe dysfunctional phenotype in CD8+ T cells in cancer. It hasalso been expressed in regulatory T cells (Treg) in certain cancers. Inview of the involvement the TIM3 pathway in key immune cell populationsthat are immunosuppressed in some cancers, it represents an attractivecandidate for immuno-oncology therapy. See, Anderson, A. C., CancerImmunol Res., (2014) 2:393-398; and Ferris, R. L., et al., J Immunol.(2014) 193:1525-1530.

A. Bispecific Binding Proteins

Adding multiple binding sites to a molecule having specificity for asingle binding domain can greatly enhance the capabilities (e.g.therapeutic, diagnostic, etc) of the molecule. For example, a bispecificantibody may bind to more than one region of the same targetbiomolecule, conferring greater specificity than a mono-specificpolypeptide that binds to only one epitope on a target. Alternately, abispecific antibody may bind to multiple target biomolecules, such astargets that are present in a complex, or targets for which sequesteringand/or clustering is desired. In a third scenario, the same bispecificantibody may perform distinct functions at any given time, depending onthe localization and/or expression of its target molecules.

Described herein are novel binding proteins. One such configuration ofthese novel binding proteins is referred to as “DuetMab.” DuetMab hasthe following basic structure: an Fc region having a modified heavychain, wherein the CH1 region of the modified heavy chain has asubstitution of a native cysteine to a non-cysteine amino acid, and asubstitution of a native non-cysteine amino acid to a cysteine aminoacid; a modified corresponding light chain, where the CL region of themodified light chain also has a substitution of a native cysteine to anon-cysteine amino acid, and a substitution of a native non-cysteineamino acid to a cysteine amino acid; a second Fc region having a secondheavy chain; and second corresponding modified light chain, where themodified heavy chain is directly linked to the corresponding modifiedlight chain, and on a separate target binding arm, the second heavychain is directly linked to the second corresponding light chain, andwhere the substituted cysteine of the modified heavy chain, resultingfrom the substitution of the native non-cysteine amino acid to thecysteine amino acid, and the substituted cysteine of the modifiedcorresponding light chain, resulting from the substitution of the nativenon-cysteine amino acid to the cysteine amino acid, can form adisulphide bond. Disclosure related to DuetMab can found e.g., in U.S.Pat. No. 9,527,927, incorporated herein by reference in its entirety.

Additional exemplary configurations of these novel binding proteins arereferred to as “BiSAb” or “BiSAbs”. Schematic representations ofexemplary BiSAbs, as well as specific examples of particular BiSAbs areprovided herein. More generally, a BiSAb is a polypeptide containing twobinding units, each of which binds to an epitope (e.g., binding unit 1binds to a first epitope and binding unit 2 binds to a second epitope).The basic BiSAb is bivalent for binding to each of the two epitopes(e.g., the polypeptide comprises two binding unit 1's (“BD1” or “BU1”)and two binding unit 2's (“BD2” or “BU2”)). Thus, where the binding unit1 and 2 bind different epitopes, the BiSAb has the multi-specificity ofa conventional bispecific antibody and the bivalency of a conventionalantibody molecule. In embodiments where binding unit 1 and 2 bind thesame epitope the BiSAb has the monospecificity of a conventionalantibody but is tetravalent. In addition to binding units, BiSAbs alsoinclude linker polypeptides and an Fc portion. The disclosure relates toa broad set of bispecific binding proteins, such as the BiSAb andproteins comprising a BiSAb core, that target molecules that modulateimmune response. Generally, the novel binding protein platforms andexemplary bispecific binding proteins (BiSAbs) described herein comprisebinding units/domains, linker polypeptides and an Fc portion. Thedisclosure also provides nucleic acid molecules encoding such BiSAbs aswell as vectors and host cells that include such nucleic acids and whichmay be used in methods of producing and using such BiSAbs. BiSAbs,binding proteins comprising a BiSAb core, and the various portions ofBiSAbs are described in greater detail herein.

In some aspects a BiSAb can comprise two heavy-light chain pairs derivedfrom a specific binding protein (i.e., antibody), wherein the heavy andlight chains each comprise a variable region (e.g. VL and VH), whichtogether form a first binding unit, and wherein the heavy chains eachfurther comprises a second binding unit (e.g. an scFv domain attached toFc or Fab). Where the first and second binding units bind differentepitopes each heavy-light chain pair is bispecific and the two pairstogether are bivalent for each epitope. Where the first and secondbinding units bind the same epitope each heavy-light chain pair ismonospecific and the two pairs together are tetravalent for the epitope.In some aspects, the two heavy-light chain pairs are identical. In someaspects, the two heavy-light chain pairs are not identical.

In specific embodiments, the domains of the BiSAbs may be based on knownimmunoglobulin domains. Immunoglobulin molecules such as monoclonalantibodies (mAbs) are widely used as diagnostic and therapeutic agents,and methods for engineering the binding fragments of mAbs are well-knownin the art. Monoclonal antibodies, like all immunoglobulin molecules,are made up of heavy chain and light chain peptide subunits, which eachinclude variable and constant domains that confer binding specificity(variable domain) and isotype (constant domain).

The BiSAbs disclosed herein may have a similar overall structure to aconventional antibody, but are distinguishable by the presence of anadditional binding unit that is attached at a location within the Fabdomain, attached at a location away from the Fab domain and within theHinge or Fc regions (e.g., within the CH2, CH3, or CH4 regions, or atthe interface of such regions such as the CH2-CH3 interface). Thus,unlike conventional antibodies that are bivalent for binding to a singleepitope, BiSAbs are bivalent for binding to two epitopes. However, asdescribed herein, BiSAbs may still maintain numerous desirableproperties of conventional antibodies, such as ability to bind FcRn andability to bind C1q and Fcγ receptors (e.g., indicative of ability tomediate antibody and complement dependent cytotoxicity).

Binding domains described herein can comprise antigen binding fragmentscontaining only portions of a mAb molecule, such as Fab, F(ab′)₂, Fab′,scFv, di-scFv, sdAb fragments, as these fragments have found use asdiagnostic or therapeutic agents. In addition, specific residues in thevariable domains can be altered to improve binding specificity and/orstability of antibodies and antibody fragments. Other residues notdirectly involved in antigen binding can be replaced in order to“humanize” regions of non-human antibodies and reduce immunogenicity ofthe mAb.

Although BiSAbs differ from conventional antibodies, for example, theyare bivalent for binding to two different epitopes (or tetravalent forbinding to a single epitope) many of the portions of BiSAbs are derivedfrom or analogous to portions of conventional antibodies. Any mAbdomains and/or fragments known in the art may be used in the BiSAbsdescribed herein. In particular, the BiSAb may comprise Fab and/or scFvfragments, or variants thereof. Exemplary, non-limiting variants of scFvinclude but are not limited to tandem di-scFvs, tandem tri-scFvs,diabodies, and tri(a)bodies.

The disclosure relates generally to novel binding proteins, of whichBiSAbs are an illustrative example. Additional examples are bindingproteins comprising a BiSAb core as well as one or more additionalbinding units and/or binding proteins comprising an extended BiSAb core.It should be understood that whenever BiSAbs or features of BiSAbs aredescribed herein, such description applies generally to the novelbinding proteins of the disclosure, regardless of whether such bindingproteins include two binding units or more than two binding units.Accordingly, the term BiSAb is exemplary of binding proteins describedherein and, where context permits, any such reference to BiSAb may alsobe used to describe binding proteins comprising a BiSAb core.

Novel BiSAb Structural Platform.

In one aspect the disclosure provides BiSAb binding proteins having astructural platform comprising domains that are generally illustrated bythe schematic diagrams in FIGS. 1A-1F. These diagrams are illustrativeand thus insertion between additional residues is also within the scopeof the disclosed binding proteins. FIGS. 1A-1C depict the Fc region ofan antibody at the CH2-CH3 interface of an IgG1 that was modeled usingPyMOL, and illustrates several exemplary BiSAbs of the disclosure. Threesurface exposed loops were identified at or near the CH2-CH3 interfacethat were likely able to withstand the insertion of a second bindingmoiety (e.g., an scFv) without compromising the structural integrity orstability of the IgG or second binding moiety. FIG. 1A is a schematicdiagram of one such representative loop ISRTP (SEQ ID NO:39) identifiedin the CH2 region near at the CH2-CH3 interface. FIG. 1D also shows arepresentative construct IS-scFv-RTP, wherein a scFv is inserted betweenS and R of the ISRTP loop. FIG. 1B is a schematic diagram of therepresentative loop AKGQP (SEQ ID NO:40) identified at the CH2-CH3interface. FIG. 1E shows a representative construct AK-scfv-GQP, whereina scFv is inserted between K and G of the AKGQP loop. FIG. 1C is aschematic diagram of the representative loop SNG identified in the CH3region downstream of the CH2-CH3 interface. FIG. 1F also shows therepresentative construct S-scfv-NG, wherein a scFv is inserted between Sand N of the SNG loop. Examples herein provide illustration ofconstructs oriented as SN-scFv-G, wherein a scFv is inserted between Nand G of the SNG loop.

Thus, one aspect of the disclosure relates to a BiSAb that comprises twoidentical heavy-light chain pairs, wherein each heavy-light chain pairis bispecific and the two identical pairs are together bivalent for eachepitope. Each heavy-light chain pair comprises a binding domain (BD)that can comprise a Fab domain that binds a first epitope (binding unit1), a second binding domain (BD2) that binds a second epitope (orbinding unit 2 that may be, for example, an scFv) and an Fc region. Insome embodiments the second binding domain may be associated with theFab domain. In some embodiments the Fc region of the BiSAb may beassociated with the second binding domain (BD2) that binds a secondepitope (binding unit 2; depicted as an scFv in FIGS. 1D-1F).

In some embodiments the disclosure provides for a BiSAb having a generalplatform structure that comprises two chimeric heavy chains, eachcomprising a heavy chain variable region (VH1), a heavy chain constantregion (CH1), a hinge or polypeptide linker region, an Fc regioncomprising a CH2 domain and a CH3 domain, wherein a second bindingdomain (BD2), optionally flanked on one or both sides by a polypeptidelinker (L1 and/or L2) is associated with solvent exposed loops in the Fcregion in the sequence of (i) the CH2 region, (ii) the interface of theCH2 and CH3 region, or (iii) the CH3 region. The BiSAb of this aspect ofthe disclosure also comprises two conventional antibody light chains,each comprising a light chain variable region (VL1) and light chainconstant region (CL), which forms part of the first binding domain(BD1). The binding domain (BD2) of the particular BiSAb illustrated inFIGS. 1D-1F is an scFv.

FIGS. 1D-1F provide a useful schematic representation of a BiSAb, whichmay also be referred to herein as a BiSAb “core”. The polypeptide chain,as shown in FIG. 1D, comprises a heavy chain having: a VH1 domain, a CH1domain, a hinge/linker, a partial N-terminal CH2 domain, an optionallinker (referred to herein as L1 or a first polypeptide linker), bindingunit 2 (such as VL2 and VH2 of an scFv), another optional linker (e.g.,L2 or a second polypeptide linker), the remaining C-terminal CH2 domain,and a CH3 domain. Because this heavy chain may include BD2 havingalternative binding proteins and/or traditional light chain regions, itis referred to herein as a chimeric heavy chain. A BiSAb comprises twosuch chimeric heavy chains, and these may be the same or different. Notethat the variable heavy chain domain (VH) for binding unit 1 is referredto as VH1. In certain aspects, this is a variable heavy chain of a Fabthat binds to a first epitope. Similarly, the variable light chaindomain (VL) for binding unit 1 is referred to as VL1. In certainaspects, this is a variable light chain of a Fab that binds to a firstepitope. In contrast, the domains for binding unit two are denoted withthe number “2”, such as VH2 and VL2 for aspects in which binding unit 2is an scFv that binds to a second epitope.

Similarly, the polypeptide chain, as shown in FIG. 1E, comprises a heavychain having: a VH1 domain, a CH1 domain, a hinge/linker, a CH2 domain,an optional linker (referred to herein as L1 or a first polypeptidelinker), binding unit 2 (such as VL2 and VH2 of an scFv), anotheroptional linker (e.g., L2 or a second polypeptide linker), and a CH3domain. In this embodiment the BiSAb comprises a second binding domain,illustrated as scFv associated with the Fc at the sequence at theinterface of the CH2 and CH3 regions.

The polypeptide chain, as shown in FIG. 1F, comprises a heavy chainhaving: a VH1 domain, a CH1 domain, a hinge/linker, a CH2 domain, apartial CH3 domain, an optional linker (referred to herein as L1 or afirst polypeptide linker), binding unit 2 (such as VL2 and VH2 of anscFv), another optional linker (e.g., L2 or a second polypeptidelinker), the CH3 domain.

In these embodiments the BiSAbs typically include a typical or modifiedantibody hinge region in the chimeric heavy chain sequences.Non-limiting examples of amino acid sequences that contain a hingeregion include: EPKSCDKTHTCPPCP (SEQ ID NO:44); EPKSCDKT (SEQ ID NO:45);EPKSCGKT (SEQ ID NO:46); EPKSC (SEQ ID NO:47).

Having described the general format for the aspects relating to theparticular structural platform for certain BiSAb molecules disclosedherein, the various portions and exemplary functional properties of thedisclosed BiSAbs are described in greater detail below. In otherembodiments, the disclosure contemplates and provides other BiSAbbinding proteins that comprise alternative structural formats andarrangements which are described briefly herein as well as in otherdisclosures (see, e.g., US Publication No. 20090155275 and U.S. Pat. No.9,580,509) each of which are incorporated herein by reference.

1. Binding Units

BiSAbs of the disclosure comprise at least two binding units or bindingdomains (binding unit/domain 1 and binding unit/domain 2). In certainaspects each binding unit binds to a different epitope, either differentepitopes located on the same target molecule or epitopes on differenttargets. Because each binding unit of a BiSAb is present as a pair(there are two binding unit 1s and two binding unit 2s) BiSAbs exhibitbivalent binding to each epitope. It will be understood from theteachings herein, that where each binding unit binds the same epitope aBiSAb will exhibit tetravalent binding to the epitope.

In certain aspects, the first binding unit is a Fab fragment, forexample, a Fab fragment of a conventional monoclonal antibody or arecombinantly produced antigen binding fragment comprising a variablelight chain (VL1), a constant light chain (CL), a variable heavy chain(VH1), and a constant heavy chain portion (CH1). Optionally, the lightand heavy chains of the Fab may be interconnected via one or moredisulfide linkages such as, for example, via a suitable antibody hingeregion. The Fab binds to a first epitope.

In certain aspects, the Fab is derived from or based on the sequence ofa conventional monoclonal antibody, such as a conventional murine,humanized, or human antibody. In certain aspects, BiSAb containing theFab derived from or based on the sequence of a conventional monoclonalantibody retains one or more functional activities of the conventionalantibody (e.g., retains at least 80% or more (80%, 85%, 90%, 95%, 97%,98%, 99% or 100%) of a functional activity). For example, in certainaspects, the BiSAb containing such a Fab retains one or more of theaffinity for antigen, inhibitory activity, immune system modulationactivity, activation or induction of an immune response, and/or cell(e.g., cancer cell) killing activity of the conventional antibody.

In certain aspects, BiSAbs of the disclosure comprise binding unit 2 andbinding unit 2 comprises a binding domain that binds a second epitope.The binding unit 2 (or binding domain 2 (BD2)) may be associated withthe BiSAb using any suitable strategy. As used herein a BD2 that is“associated” with the BiSAb (e.g., within the Fc region in someembodiments, within the Fab region in other embodiments) means that thetwo molecules have an interaction between them such that the BD2 retainsorientation for target binding and association with the Fc portion orwith the Fab portion of the BiSAb structure. Examples of suchinteractions include covalent bonding via an amino acid linkers,covalent bonding through recombinant expression of BD2 within the Fabregion, within the hinge region, or within the Fc region at the CH2,CH3, or interface of CH2 and CH3, or the CH4 region, and non-covalentinteractions such as van der Waals and hydrogen bonding interactionswithin those same regions. Non-limiting examples of binding domains (or“BDs” or “binding units”) falling within the scope of the disclosureinclude antibody variable regions, antibody fragments, scFvs, singlechain diabodies, or other binding domains known in the art. Bindingdomains also include bispecific single chain diabodies, or single chaindiabodies designed to bind two distinct epitopes In one aspect, epitopebinding domains useful in the construction of multispecific epitopebinding domains of the disclosure are exemplified in US20100298541 andUS20130079280 which are hereby incorporated by reference for allpurposes.

In certain aspects, the BiSAb can comprise a binding domain thatincludes an scFv. Thus, in certain aspects, binding unit 2 comprises anscFv. It is to be understood that an scFv encompasses a polypeptidechain comprising a variable heavy chain domain (VH) linked to a variablelight chain domain (VL) via a flexible polypeptide linker. FIGS. 1D-1Fshow a schematic of an exemplary BiSAb, wherein the BD (here, depictedas binding unit 2) is an scFv having domains as described herein thatmay be designated as VL2 and VH2. In some aspects the polypeptide linkerbetween VH2 and VL2 comprises a protease cleavage site. The VH and VLdomains of the scFv may be derived from the same or from differentantibodies. In some aspects, a VH or VL of the scFv may comprise one ormore CDRs which bind to a target of interest, while the remainder of theVH or VL domain is derived from a different antibody or is synthetic. Insome aspects, the scFv comprises at least one CDR of an antibody, e.g.,an antibody known in the art to bind to a target of interest. In someaspects, the scFv comprises at least two CDRs of a given antibody. Insome aspects, the scFv comprises at least three CDRs of a givenantibody. In some aspects, the scFv comprises at least four CDRs of agiven antibody. In some aspects, the scFv comprises at least five CDRsof a given antibody. In some aspects, the scFv comprises at least sixCDRs of a given antibody.

In certain aspects, the BD may comprise a ligand binding domain of areceptor or a receptor binding domain of a ligand. In some aspects theBD comprises a sequence that has binding affinity for one or moreepitopes on a target selected from the group consisting of CTLA-4, PD-1,PD-L1, OX40, and TIM3, as described above. In some embodiments thebinding domain exhibits specific binding activity for a target selectedfrom the group consisting of CTLA-4, PD-1, PD-L1, OX40, and TIM3. TheBiSAbs disclosed herein can comprise any combination of binding domainsthat have binding affinity or specific binding activity for themolecular targets disclosed herein. For example the BiSAbs disclosedherein may comprise a combination of binding domains that allow forbispecific binding to targets including; CTLA-4 and PD-1; CTLA-4 andPD-L1; CTLA-4 and TIM3; PD-1 and PD-L1; PD-L1 and OX40; PD-1 and TIM3;PD-L1 and TIM3; and TIM3. BiSAbs that include binding domains that bindparticular target combinations are illustrated in the Examples andinclude the non-limiting combinations of PD-1/CTLA-4; PD-L1/CTLA-4;PD-1/OX40; PD-L1/OX40; and PD-1/TIM3.

In some further embodiments, the BiSAbs exhibit a binding activity(e.g., binding affinity and/or binding specificity) for at least one ofthe target molecules that is greater the binding activity of theparental monospecific binding sequence used to generate the BiSAb. Insimilar embodiments, the BiSAbs can exhibit a binding activity (e.g.,binding affinity and/or binding specificity) for both of the targetmolecules that is greater than the binding activity of both of theparental monospecific binding sequences used to generate the BiSAbs. Inyet a further embodiment, the BiSAbs can exhibit a binding activity(e.g., binding affinity and/or binding specificity) for both of thetarget molecules that is greater than the binding activity for thecombination of the parental monospecific binding sequences used togenerate the BiSAbs. The enhancement of the binding properties of theBiSAbs relative to the parental monospecific binding sequences, eitheralone or in combination, provide unexpected advantages relative to theuse of monospecific therapeutics that target the same molecules, evenwhen used in combination.

In some embodiments the disclosure relates an antibody, or antigenbinding fragment thereof, that binds to a target selected from the groupconsisting of CTLA-4, PD-1, PD-L1, OX40, and TIM3. In such embodimentsthe antibody, or antigen binding fragment thereof, may comprise a heavychain sequence and a light chain sequence, or a portion of a heavy chainsequence and a light chain sequence that comprises the CDR1, CDR2, andCDR3 sequences of the heavy and the light chain sequences. In otherembodiments the antibody, or antigen binding fragment thereof, maycomprise a heavy chain variable (HCv) region sequence and a light chainvariable (LCv) region sequence, or a portion of a HCv and a LCv thatcomprises the CDR1, CDR2, and CDR3 sequences of the heavy and the lightchain sequences. In yet other embodiments the antibody, or antigenbinding fragment thereof, may comprise the CDR1, CDR2, and CDR3sequences of the heavy and the light chain sequences. In someembodiments the antibody may be a chimeric, a humanized, or a humanantibody. In some embodiments the antibody may be a polyclonal or amonoclonal antibody. In further embodiments, the antibody is amonoclonal antibody.

In some embodiments, domains that comprise all or a portion of anantigen binding region of such “parental” antibodies as discussed abovemay be used to generate the bispecific binding proteins (BiSAbs) thatare disclosed herein. The non-limiting embodiments that are illustratedin the Examples provide a description relating to how antibody sequencesmay be identified and combined to produce a BiSAb that exhibitsbispecific binding to a combination of molecular targets.

Several methodologies can be used alone or in combination to improve thestability of a BiSAb comprising an scFv molecule. One potentialmethodology that can be used, alone or in combination with one or moreof the other methodologies described herein, is engineered the lengthand/or composition of the linker connecting the scFv domains tostabilize the scFv portion.

Another potential methodology that can be used is to introduce at leasttwo amino acid substitutions (also referred to as modifications ormutations) into the VH and/or VL domains of the scFv so as to promotedisulfide bond formation (see for example Brinkmann et al., 1993, PNAS,90:7538-42; Zhu et al., 1997, Prot. Sci. 6:781-8; Reiter et al., 1994,Biochem. 33:5451-9; Reiter et al., 1996, Nature 14: 1239-45; Luo et al.,1995, J. Biochem. 118:825-31; Young et al., 1995, FEBS Let. 377:135-9;Glockshuber et al., 1990, Biochem. 29:1362-7). This method can be usedalone or in combination with one or more of the other methodologiesdescribed herein.

In certain aspects, one or more mutations can be introduced into each ofthe VH and VL domains of the scFv to promote interchain disulfide bondformation between the VH and VL domains upon expression of a BiSAbcomprising an scFv. In another aspect, the two mutations are introducedin the same domain of the chain. In a certain aspect, the two mutationsare introduced in different chains. In certain aspects, multiplecomplementary mutations are introduced to promote formation of multipledisulfide bonds or other stabilizing interactions. In certain aspects, acysteine is introduced to promote the disulfide bond formation.Exemplary amino acids that may be mutated to cysteine include aminoacids 43, 44, 45, 46, 47, 103, 104, 105, and 106 of VH2 and amino acids42, 43, 44, 45, 46, 98, 99, 100, and 101 of VL2. The foregoing numberingis based on Kabat numbering identifying the position relative only tothe VH2 and VL2 of the scFv (and not relative to the position of theamino acid in the full length sequence of the BiSAb or SEQ ID NOsprovided herein). Exemplary combinations of amino acid positions whichmay be mutated to cysteine residues include: VH44-VL100, VH105-VL43,VH105-VL42, VH44-VL101, VH106-VL43, VH104-VL43, VH44-VL99, VH45-VL98,VH46-VL98, VH103-VL43, VH103-VL44, and VH103-VL45. In some aspects,amino acid 44 of VH and amino acid 100 of VL are mutated to cysteines.

Another method that can be used, alone or in combination with one ormore of the other methods described herein, is selecting the order ofthe domains of the scFv. In certain aspects, the orientation of the VHdomain relative to the VL domain is optimized for stability. In certainaspects, the scFv is in the VH-linker-VL orientation. In certainaspects, the scFv is in the VL-linker-VH orientation. In embodimentsrelating to the novel BiSAb format disclosed herein, the orientation ofthe domains in the scFv can determine how the scFv associates with theFc portion of the BiSAb. While this is described in more detail below inthe context of polypeptide linkers. Briefly, however, given that the BD(e.g., an scFv) is interconnected to the CH2, CH3, or at the interfaceof CH2 and CH3 by optional polypeptide linkers (L1) and (L2), the orderof domains determines which portion of the scFv is interconnected to L1and which portion of the scFv is interconnected to L2.

A further method that can be used, alone or in combination with theother methods described herein, is to introduce one or more stabilizingmutations by mutating one or more surface residues of the scFv. In someaspects, one, two, three, four, five, six, or more than six residues aremutated in one or both of the VH and/or VL domain of the scFv. Incertain aspects, changes are made in only the VH domain of the scFv. Incertain aspects, changes are made in only the VL domain of the scFv. Incertain aspects, changes are made in both the VH and VL domains of thescFv. The same number of changes may be made in each domain or adifferent number of changes may be made in each domain. In certainaspects, one or more of the changes is a conservative amino acidsubstitution from the residue present in the unmodified, parent scFv. Inother aspects, one or more of the changes is a non-conservative aminoacid substitution from the residue present in the unmodified, parentscFv. When multiple substitutions are made, either in one or both of theVH or VL domains of the scFv, each substitution is independently aconservative or a non-conservative substitution. In certain aspects, allof the substitutions are conservative substitutions. In certain aspects,all of the substitutions are non-conservative. In certain aspects, atleast one of the substitutions is conservative. In certain aspects, atleast one or the substitutions is non-conservative.

Yet another method that can be used, on its own or in combination withother methods, is to introduce one or more amino acid substitutions bymutating one or more residues present in the VH and/or VL domain of thescFv to match the most frequent residue at said particular position of aconsensus sequence of VH and/or VL domain of known antibodies. Incertain aspects, substitutions are introduced at one, two, three, four,five, six, or more than six positions in one or both of the VH domainand/or the VL domain of the scFv. The same number of changes may be madein each domain or a different number of changes may be made in eachdomain. In certain aspects, one or more of the changes in sequence matchthat of a given consensus is a conservative amino acid substitution fromthe residue present in the unmodified VH and/or VL sequence. In otheraspects, one or more of the changes represent a non-conservative aminoacid substitution from the residue present in the unmodified VH and/orVL sequence. When multiple substitutions are made, either in one or bothof the VH or VL domain of the scFv, each substitution is independently aconservative or a non-conservative substitution. In certain aspects, allof the substitutions are conservative substitutions. In certain aspects,all of the substitutions are non-conservative substitutions. In certainaspects, at least one of the substitutions is conservative. In certainaspects, at least one or the substitutions is non-conservative.

It should be noted that any of the modifications described as useful formodifying or stabilizing the scFv portion can be applied to modify theFab portion. For example, the variable domains of the Fab portion of aBiSAb can be modified to improve stability, antigen binding and thelike. Moreover, either the Fab or scFv portion can be modified to reduceimmunogenicity.

In certain aspects, binding unit 2 (the BD) is an scFv, for example, anscFv derived from a conventional monoclonal antibody comprising avariable light chain (VL2) and a variable heavy chain (VH2)interconnected by a flexible linker, such as a glycine-serine linker.Optionally, the variable light and variable heavy chains of the scFv maybe further interconnected via one or more disulfide linkages, and asdescribed above, may include one or more mutations or variations. ThescFv binds to a second epitope. In certain aspects the second epitope isdifferent from the first epitope bound by binding unit 1. In otheraspects the second epitope is the same as the first epitope bound bybinding unit 1. In certain aspects, the scFv is derived from or based onthe sequence of a conventional monoclonal antibody, such as aconventional murine, humanized or human antibody. In certain aspects,BiSAb containing the scFv derived from or based on the sequence of aconventional monoclonal antibody retains one or more functionalactivities of the conventional antibody (e.g., retains at least 80% ormore (80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) of a functionalactivity). For example, in certain aspects, the BiSAb containing such anscFv retains one or more of the affinity for antigen, inhibitoryactivity, or cell killing activity of the conventional antibody.

In certain aspects a BiSAb comprises any of the binding unit is andbinding unit 2s described herein, including any combination of a bindingunit 1 and a binding unit 2. For example, in certain aspects, thedisclosure provides a polypeptide comprising a Fab that binds to aparticular target (e.g., that binds to an epitope on a particulartarget), such as a Fab comprising a particular amino acid sequence orencoded by a particular nucleotide sequence and/or an scFv that binds toa particular target (e.g., that binds to an epitope on a particulartarget), such as an scFv comprising a particular amino acid sequence orencoded by a particular nucleotide sequence.

As described in detail above, binding unit 1 and binding unit 2 may beassociated with the BiSAb via covalent bonding via a linker polypeptide1 (L1, L2). Generally, the linkage is via the chimeric heavy chain ofthe BiSAb, such that the interconnection is via the heavy chain C_(H)2domain, the heavy chain C_(H)3 domain, or at the interface of the heavychain C_(H)2 domain and C_(H)3 domain or, in some embodiments, withinthe hinge region or Fab domain. L1 and L2 can vary in length andsequence independently from each other, and exemplary configurations aredescribed herein. The disclosure contemplates BiSAbs comprising anycombination of binding units and linker polypeptides, including anycombination of the specific binding units that bind desired target(s)and specific L1 and L2 polypeptide linkers described herein.

2. Fc Region

As used herein, “Fc region” encompasses domains derived from theconstant region of an immunoglobulin, preferably a human immunoglobulin,including a fragment, analog, variant, mutant or derivative of theconstant region. Suitable immunoglobulins include IgG1, IgG2, IgG3,IgG4, and other classes such as IgA, IgD, IgE and IgM. The Fc region maybe a native sequence Fc region or an altered Fc region. The Fc region ofan immunoglobulin generally comprises two constant domains, a C_(H)2domain and a C_(H)3 domain, and optionally comprises a C_(H)4 domain.BiSAbs of the disclosure include an Fc region comprising a C_(H)2 domainand a C_(H)3 domain.

a. Altered Fc Regions

Altered Fc regions (also referred to herein as “variant Fc regions”) maybe used to alter the effector function and/or half-life of a BiSAb ofthe disclosure. One or more alterations may be made in the Fc region inorder to change functional and/or pharmacokinetic properties ofmolecules. Such alterations may result in a decrease or increase of C1qbinding and complement dependent cytotoxicity (CDC) or of FcγR binding,for IgG, and antibody-dependent cellular cytotoxicity (ADCC), orantibody dependent cell-mediated phagocytosis (ADCP). The presentdisclosure encompasses BiSAbs wherein changes have been made to finetune the effector function, either by enhancing or diminishing functionor providing a desired effector function. Accordingly, in one aspect ofthe disclosure, the BiSAbs comprise a variant Fc region (i.e., Fcregions that have been altered as discussed below). BiSAbs comprising avariant Fc region are also referred to here as “Fc variant BiSAbs.” Asused herein “native” refers to the unmodified parental sequence and theBiSAb comprising a native Fc region is herein referred to as a “nativeFc BiSAb”. Fc variant BiSAbs can be generated by numerous methods wellknown to one skilled in the art. Non-limiting examples include,isolating antibody coding regions (e.g., from hybridoma) and making oneor more desired substitutions in the Fc region. Alternatively, theantigen-binding portion (e.g., variable regions) of a BiSAb may besubcloned into a vector encoding a variant Fc region. In one aspect, thevariant Fc region exhibits a similar level of inducing effector functionas compared to the native Fc region. In another aspect, the variant Fcregion exhibits a higher induction of effector function as compared tothe native Fc. In another aspect, the variant Fc region exhibits lowerinduction of effector function as compared to the native Fc. Somespecific aspects of variant Fc regions are detailed infra. Methods formeasuring effector function are well known in the art.

In general, the effector function is modified through changes in the Fcregion, including but not limited to, amino acid substitutions, aminoacid additions, amino acid deletions and changes in post translationalmodifications to Fc amino acids (e.g. glycosylation). The methodsdescribed below may be used to fine tune the effector function of aBiSAb of the disclosure, a ratio of the binding properties of the Fcregion for the FcR (e.g., affinity and specificity), resulting in aBiSAb with the desired properties.

It is understood that the Fc region as used herein includes thepolypeptides comprising the constant region of an antibody molecule,excluding the first constant region immunoglobulin domain. Thus Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, and the last three constant region immunoglobulin domainsof IgE and IgM, and, optionally, all or a portion of the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3(Cγ2 and Cγ3) and optionally a portion of the lower hinge betweenCgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fcregion may vary, as used herein the human IgG heavy chain Fc regioncomprises residues A231 to its carboxyl-terminus, wherein the numberingis according to the EU index as set forth in Kabat. Fc may refer to thisregion in isolation, or this region in the context of an antibody,antibody fragment, or Fc fusion protein. Polymorphisms have beenobserved at a number of different Fc positions, including but notlimited to positions 270, 272, 312, 315, 356, and 358 of IgG1 asnumbered by the EU index, and thus slight differences between thepresented sequence and sequences in the prior art may exist.

In one aspect, the present disclosure encompasses Fc variant BiSAbswhich have altered binding properties for an Fc ligand (e.g., an Fcreceptor, C1q) relative to a native Fc BiSAb. Examples of bindingproperties include but are not limited to, binding specificity,equilibrium dissociation constant (K_(d)), dissociation and associationrates (k_(off) and k_(on) respectively), binding affinity and/oravidity. It is known in the art that the equilibrium dissociationconstant (K_(d)) is defined as k_(off)/k_(on). In certain aspects, aBiSAb comprising an Fc variant region with a low K_(d) may be moredesirable than a BiSAb with a high K_(d). However, in some instances thevalue of the k_(on) or k_(off) may be more relevant than the value ofthe K_(d). One skilled in the art can determine which kinetic parameteris most important for a given application. For example, a modificationthat reduces binding to one or more positive regulator (e.g., FcγRIIIA)and/or enhanced binding to an inhibitory Fc receptor (e.g., FcγRIIB)would be suitable for reducing ADCC activity. Accordingly, the ratio ofbinding affinities (e.g., the ratio of equilibrium dissociationconstants (K_(d))) for different receptors can indicate if the ADCCactivity of an Fc variant BiSAb of the disclosure is enhanced ordecreased. Additionally, a modification that reduces binding to C1qwould be suitable for reducing or eliminating CDC activity.

In one aspect, Fc variant BiSAbs exhibit altered binding affinity forone or more Fc receptors including, but not limited to FcRn, FcγRI(CD64) including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32including isoforms FcγRIIA, FcγRIIB, and FcγRIIC); and FcγRIII (CD16,including isoforms FcγRIIIA and FcγRIIIB) as compared to a native FcBiSAb.

In certain aspects, an Fc variant BiSAb has increased affinity for an Fcligand. In other aspects, an Fc variant BiSAb has decreased affinity foran Fc ligand relative to a native Fc BiSAb.

In a specific aspect, an Fc variant BiSAb has enhanced binding to the Fcreceptor FcγRIIIA In another specific aspect, an Fc variant BiSAb hasenhanced binding to the Fc receptor FcγRIIB In a further specificaspect, an Fc variant BiSAb has enhanced binding to both the Fcreceptors FcγRIIIA and FcγRIIB In certain aspects, Fc variant BiSAbsthat have enhanced binding to FcγRIIIA do not have a concomitantincrease in binding the FcγRIIB receptor as compared to a native FcBiSAb. In a specific aspect, an Fc variant BiSAb has reduced binding tothe Fc receptor FcγRIIIA In a further specific aspect, an Fc variantBiSAb has reduced binding to the Fc receptor FcγRIIB In another specificaspect, and Fc variant BiSAb has enhanced binding to the Fc receptorFcRn. In still another specific aspect, an Fc variant BiSAb exhibitingaltered affinity for FcγRIIIA and/or FcγRIIB has enhanced binding to theFc receptor FcRn. In yet another specific aspect, an Fc variant BiSAbexhibiting altered affinity for FcγRIIIA and/or FcγRIIB has alteredbinding to C1q relative to a native Fc BiSAb.

In another aspect, Fc variant BiSAbs exhibit increased or decreasedaffinities to C1q relative to a native Fc BiSAb. In still anotherspecific aspect, an Fc variant BiSAb exhibiting altered affinity for C1qhas enhanced binding to the Fc receptor FcRn. In yet another specificaspect, an Fc variant BiSAb exhibiting altered affinity for C1q hasaltered binding to FcγRIIIA and/or FcγRIIB relative to a native FcBiSAb.

It is recognized that antibodies are capable of directing the attack anddestruction of targeted antigen through multiple processes collectivelyknown in the art as antibody effector functions. One of these processes,known as “antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a form of cytotoxicity in which secreted Ig bound onto Fcgamma receptors (FcγRs) present on certain cytotoxic cells (e.g.,Natural Killer (NK) cells, neutrophils, and macrophages) enables thesecytotoxic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently kill the target cell with cytotoxins.Specific high-affinity IgG antibodies directed to the surface of targetcells “arm” the cytotoxic cells and are required for such killing. Lysisof the target cell is extracellular, requires direct cell-to-cellcontact, and does not involve complement. Another process encompassed bythe term effector function is complement-dependent cytotoxicity(hereinafter referred to as “CDC”) which refers to a biochemical eventof antibody-mediated target cell destruction by the complement system.The complement system is a complex system of proteins found in normalblood plasma that combines with antibodies to destroy pathogenicbacteria and other foreign cells. Still another process encompassed bythe term effector function is antibody dependent cell-mediatedphagocytosis (ADCP) which refers to a cell-mediated reaction whereinnonspecific cytotoxic cells that express one or more effector ligandsrecognize bound antibody on a target cell and subsequently causephagocytosis of the target cell.

It is contemplated that Fc variant BiSAbs are characterized by in vitrofunctional assays for determining one or more FcγR mediated effectorcell functions. In certain aspects, Fc variant BiSAbs have similarbinding properties and effector cell functions in in vivo models (suchas those described and disclosed herein) as those in in vitro basedassays. However, the present disclosure does not exclude Fc variantBiSAbs that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. The term“antibody half-life” as used herein means a pharmacokinetic property ofan antibody that is a measure of the mean survival time of antibodymolecules following their administration. Antibody half-life can beexpressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body (or other mammal) ora specific compartment thereof, for example, as measured in serum, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody (or BiSAb) half-life results in an increase in meanresidence time (MRT) in circulation for the BiSAb administered.

The increase in half-life allows for the reduction in amount of druggiven to a patient as well as reducing the frequency of administration.To increase the serum half-life of a BiSAb, one may incorporate asalvage receptor binding epitope into the BiSAb (especially an antibodyfragment) as described in U.S. Pat. No. 5,739,277, for example. As usedherein, the term “salvage receptor binding epitope” refers to an epitopeof the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the in vivo serum half-life of theIgG molecule. Alternatively, BiSAbs of the disclosure with increasedhalf-lives may be generated by modifying amino acid residues identifiedas involved in the interaction between the Fc and the FcRn receptor(see, for examples, U.S. Pat. Nos. 6,821,505 and 7,083,784). Inaddition, the half-life of BiSAbs of the disclosure may be increased byconjugation to PEG or albumin by techniques widely utilized in the art.

It is contemplated that either insertion of additional binding domainsinto the Fc region as described here and/or subsequent binding byantigen may affect Fc activity. For instance, binding antigen mayincrease or decrease binding affinity and activity for FcgRs, C1q, andFcRn. This would create an antigen-dependent switch to modulate variousantibody-dependent processes. In one aspect, antigen binding maydecrease interaction with FcRn, allowing a free BiSAb to interact withFcRn and have a normal half-life, but allow rapid clearance/cellularinternalization of BiSAb-Ag complexes. Further, this could allowBD2-antigen mediated interactions to have an effect on the clearance ofantigens bound by BD1. In an additional aspect, the BiSAb could comprisethe Fc region directly inserted to BD2 (Fc-BD2).

In one aspect, the present disclosure provides Fc variants, wherein theFc region comprises a modification (e.g., amino acid substitutions,amino acid insertions, amino acid deletions) at one or more positionsselected from the group consisting of 221, 225, 228, 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256,257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416,419, 421, 428, 433, 434, 435, 436, 440, and 443 as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may comprise amodification at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 7,083,784; 7,317,091; 7,217,797; 7,276,585; 7,355,008).Additional, useful amino acid positions and specific substitutions areexemplified in Tables 2, and 6-10 of U.S. Pat. No. 6,737,056; the tablespresented in FIG. 41 of US 2006/024298; the tables presented in FIGS. 5,12, and 15 of US 2006/235208; the tables presented in FIGS. 8, 9 and 10of US 2006/0173170 and the tables presented in FIGS. 8-10, 13 and 14 ofWO 09/058492.

In a specific aspect, the present disclosure provides an Fc variant,wherein the Fc region comprises at least one substitution selected fromthe group consisting of 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E,234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W,235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235E, 235F, 236E,237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I,240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R. 243W, 243L 243Y, 243R,243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S,254T, 255L, 256E, 256F, 256M, 257C, 257M, 257N, 2621, 262A, 262T, 262E,263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y,264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T,266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E,280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I,296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L,299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F313F, 316D, 318A, 318S,320A, 320S, 322A, 322S, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V,325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 328S,328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F,329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R,330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V,331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F,332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S,333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 424F,434W, 434Y, 436H, 440Y and 443W as numbered by the EU index as set forthin Kabat. Optionally, the Fc region may comprise additional and/oralternative amino acid substitutions known to one skilled in the artincluding, but not limited to, those exemplified in Tables 2, and 6-10of U.S. Pat. No. 6,737,056; the tables presented in FIG. 41 of US2006/024298; the tables presented in FIGS. 5, 12, and 15 of US2006/235208; the tables presented in FIGS. 8, 9 and 10 of US2006/0173170 and the tables presented in FIGS. 8, 9 and 10 ofUS20090041770, all of which are incorporated herein by reference.

In a specific aspect, the disclosure provides an Fc variant BiSAb,wherein the Fc region comprises at least one modification (e.g., aminoacid substitutions, amino acid insertions, amino acid deletions) at oneor more positions selected from the group consisting of 228, 234, 235and 331 as numbered by the EU index as set forth in Kabat. In oneaspect, the modification is at least one substitution selected from thegroup consisting of 228P, 234F, 235E, 235F, 235Y, and 331S as numberedby the EU index as set forth in Kabat.

In another specific aspect, the present disclosure provides an Fcvariant BiSAb, wherein the Fc region is an IgG4 Fc region and comprisesat least one modification at one or more positions selected from thegroup consisting of 228 and 235 as numbered by the EU index as set forthin Kabat. In still another specific aspect, the Fc region is an IgG4 Fcregion and the non-naturally occurring amino acids are selected from thegroup consisting of 228P, 235E and 235Y as numbered by the EU index asset forth in Kabat.

In another specific aspect, the present disclosure provides an Fcvariant BiSAb, wherein the Fc region comprises at least onenon-naturally occurring amino acid at one or more positions selectedfrom the group consisting of 239, 330 and 332 as numbered by the EUindex as set forth in Kabat. In one aspect, the modification is at leastone substitution selected from the group consisting of 239D, 330L, 330Y,and 332E as numbered by the EU index as set forth in Kabat. See, U.S.Pat. No. 7,317,091, incorporated herein by referenced in its entirety.

In a specific aspect, the present disclosure provides an Fc variantBiSAb, wherein the Fc region comprises at least one non-naturallyoccurring amino acid at one or more positions selected from the groupconsisting of 252, 254, and 256 as numbered by the EU index as set forthin Kabat. In one aspect, the modification is at least one substitutionselected from the group consisting of 252Y, 254T and 256E as numbered bythe EU index as set forth in Kabat. See, U.S. Pat. No. 7,083,784,incorporated herein by reference in its entirety.

In certain aspects, the present disclosure provides an Fc variant BiSAb,wherein the Fc region comprises a non-naturally occurring amino acid atposition 428 as numbered by the EU index as set forth in Kabat. In oneaspect, the modification at position 428 is selected from the groupconsisting of 428T, 428L, 428F, and 428S as numbered by the EU index asset forth in Kabat. See, U.S. Pat. No. 7,670,600, incorporated herein byreference in its entirety. In another aspect, an Fc variant BiSAb mayfurther comprises a non-naturally occurring amino acid at position 434as numbered by the EU index as set forth in Kabat. In one aspect, themodification at position 434 is selected from the group consisting of434A, 434S, and 434F as numbered by the EU index as set forth in Kabat.In other aspects, the present disclosure provides an Fc variant BiSAb,wherein the Fc region comprises a non-naturally occurring amino acid atpositions 428 and 434 as numbered by the EU index as set forth in Kabat.In a specific aspect, the Fc region comprises 428L, 434S. See, U.S. Pat.No. 8,088,376.

In certain aspects, the effector functions elicited by IgG antibodiesstrongly depend on the carbohydrate moiety linked to the Fc region ofthe protein (Claudia Ferrara et al., 2006, Biotechnology andBioengineering 93:851-861). Thus, glycosylation of the Fc region can bemodified to increase or decrease effector function (see for examples,Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775; 7,393,683;7,425,446; 7,504,256; POTELLIGENT™ technology (Biowa, Inc. Princeton,N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland)). Accordingly, in one aspect theFc regions of BiSAbs of the disclosure comprise altered glycosylation ofamino acid residues. In another aspect, the altered glycosylation of theamino acid residues results in lowered effector function. In anotheraspect, the altered glycosylation of the amino acid residues results inincreased effector function. In a specific aspect, the Fc region hasreduced fucosylation. In another aspect, the Fc region is afucosylated(see for examples, U.S. Patent Application Publication No.2005/0226867). In one aspect, these BiSAbs with increased effectorfunction, specifically ADCC, are generated in host cells (e.g., CHOcells, Lemna minor) engineered to produce highly defucosylatedpolypeptide with over 100-fold higher ADCC compared to polypeptideproduced by the parental cells (Mori et al., 2004, Biotechnol Bioeng88:901-908; Cox et al., 2006, Nat Biotechnol., 24:1591-7).

Addition of sialic acid to the oligosaccharides on IgG molecules canenhance their anti-inflammatory activity and alter their cytotoxicity(Keneko et al., Science, 2006, 313:670-673; Scallon et al., Mol. Immuno.2007 March; 44(7):1524-34). The studies referenced above demonstratethat IgG molecules with increased sialylation have anti-inflammatoryproperties whereas IgG molecules with reduced sialylation have increasedimmunostimulatory properties (e.g., increase ADCC activity). Therefore,a BiSAb can be modified with an appropriate sialylation profile for aparticular application (US Publication No. 2009/0004179 andInternational Publication No. WO 2007/005786).

In one aspect, the Fc regions of BiSAbs of the disclosure comprise analtered sialylation profile compared to the native Fc region. In oneaspect, the Fc regions of BiSAbs of the disclosure comprise an increasedsialylation profile compared to the native Fc region. In another aspect,the Fc regions of BiSAbs of the disclosure comprise a decreasedsialylation profile compared to the native Fc region.

In one aspect, the Fc variants of the present disclosure may be combinedwith other known Fc variants such as those disclosed in Ghetie et al.,1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564;Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, MolImmunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543;Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferiset al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al,1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy etal, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields etal., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Pat.Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046;6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056;7,122,637; 7,183,387; 7,332,581; 7,335,742; 7,371,826; 6,821,505;6,180,377; 7,317,091; 7,355,008. Other modifications and/orsubstitutions and/or additions and/or deletions of the Fc domain will bereadily apparent to one skilled in the art.

It is notable that polypeptides presented in the BiSAb format comprisinga native Fc retain the ability to bind FcRn and C1q and to mediate ADCC,as shown in the examples. Thus, in certain aspects, a BiSAb retains theability to bind FcRn and/or C1q and/or one or more Fcgamma receptors(FcγRs). For example, in certain aspects, a BiSAb retains at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the ability tobind FcRn and/or C1q and/or one or more FcγRs, as compared to aconventional antibody that binds to one of the epitopes to which theBiSAb binds. In certain aspects, a BiSAb is generated from the bindingdomains of one or two conventional antibodies, and the comparison ofactivity is made to one or both of those conventional antibodies.

Altered Fc regions may also be used to generate heavy chainheterodimers, resulting in BiSAbs comprising two different heavy-lightchain pairs. To facilitate the formation of heterodimers the interfacebetween a pair of Fc regions is engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Incertain aspects, the interface comprises at least a part of the CH3domain. In this method, a “protrusion” is generated by replacing one ormore, small amino acid side chains from the interface of the firstantibody molecule with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers. CH3modifications include, for example, Y407V/T366S/L368A on one heavy chainand T366W on the other heavy chain; S354C/T366W on one heavy chain andY349C/Y407V/T366S/L368A on the other heavy chain. Additionalmodifications resulting in a protrusion on one chain and a cavity on theother are described in U.S. Pat. No. 7,183,076; US 2014/0348839; andMerchant et al., 1998, Nat. Biotech 16:677-681. Some non-limitingexamples of modifications that can result in a protrusion-cavityarrangement are presented in Table 1a. Other modifications which may beused to generate heterodimers include but are not limited to those whichalter the charge polarity across the Fc dimer interface such thatco-expression of electrostatically matched Fc regions results inheterodimerization. Modifications which alter the charge polarityinclude, but are not limited to, those presented in Table 1b below (alsosee, US20090182127; Gunasekaran et al., 2010, JBC 285:19637-46). Inaddition, Davis et al. (2010, Prot. Eng. Design & Selection 23:195-202)describe a heterodimeric Fc platform using strand-exchanged engineereddomain (SEED) CH3 regions which are derivatives of human IgG and IgA CH3domains (also, see WO 2007/110205).

TABLE 1a CH3 modifications for heterodimerization (protrusion-cavity)Modification(s) in one heavy chain Modification(s) in other heavy chainT366Y Y407T T366W Y407A T366Y Y407T T394W F405A T366Y/F405A T394W/Y407TT366W/F405W T394S/Y407A F405W T394S D399C K392C T366W T366S/L368A/Y407VT366W/D399C T366S/L368A/K392C/Y407V T366W/K392C T366S/D399C/L368A/Y407VS354C/T366W Y349C/T366S//L368A/Y407V Y349C/T366WS354C/T366S//L368A/Y407V E356C/T366W Y349C/T366S//L368A/Y407VY349C/T366W E356C/T366S//L368A/Y407V E357C/T366WY349C/T366S//L368A/Y407V Y349C/T366W E357C/T366S//L368A/Y407V

TABLE 1b CH3 modifications for heterodimerization Modification(s) in oneheavy chain Modification(s) in other heavy chain K370E/D399K/K439DD356K/E357K/K409D K409D D399K K409E D399K K409E D399R K409D D399R D339KE356K D399K/E356K K409D/K392D D399K/E356K K409D/K439D D399K/E357KK409D/K370D D399K/E356K/E357K K409D/K392D/K370D D399K/E357K K409D/K392DK392D/K409D D399K K409D/K360D D399K

A person skilled in the art would understand that in some aspects, an FcFusion protein can form dimers due to the homodimeric nature ofmolecules comprising an Fc region. In some aspects the Fc regions of abinding protein (e.g., BiSAb) may be differentially engineered withmutations to: promote and/or maintain heterodimerization (e.g., chimericmutations, complementary mutations, dock and lock mutations, knobs intoholes mutations, strand-exchange engineered domain (SEED) mutations,etc., see for example, U.S. Pat. No. 7,183,076; Merchant et al. (1998)Nat. Biotech 16:677-681; Ridgway et al. (1996) Protein Engineering9:617-621; Davis et al. (2010) Prot. Eng. Design & Selection 23:195-202;WO 2007/110205; WO 2007/147901; Gunasekaran et al. (2010) JBC285:19637-46, all incorporated herein by reference). Accordingly, abinding protein can be engineered to form a heterodimer comprising forexample a first binding protein, binding domain, or BiSAb fused to afirst Fc region or fragment thereof, and a second (i.e., different)binding protein, binding domain, or BiSAb fused to a second Fc region orfragment, wherein the first and second Fc regions, or fragments thereofhave been engineered to heterodimerize.

3. Glycosylation

In addition to the ability of glycosylation to alter the effectorfunction of polypeptides, modified glycosylation in the variable regioncan alter the affinity of the antibody (or BiSAb) for a target antigen.In one aspect, the glycosylation pattern in the variable region of thepresent BiSAbs is modified. For example, an aglycosylated BiSAb can bemade (i.e., the BiSAb lacks glycosylation). Glycosylation can be alteredto, for example, increase the affinity of the BiSAb for a targetantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the BiSAbsequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the BiSAb for antigen.Such an approach is described in further detail in U.S. Pat. Nos.5,714,350 and 6,350,861. One or more amino acid substitutions can alsobe made that result in elimination of a glycosylation site present inthe Fc region (e.g., Asparagine 297 of IgG). Furthermore, aglycosylatedBiSAbs may be produced in bacterial cells which lack the necessaryglycosylation machinery.

4. Polypeptide Linkers

Linkers may be used to join domains/regions of the BiSAb chimeric heavychain into a contiguous molecule. As described herein, a BiSAb mayinclude one, two, or more linker polypeptides, (e.g., L1 and L2).Additionally, a BiSAb may include additional linkers, such as a flexiblelinker interconnecting the variable heavy and light chains of an scFv.Additionally, a BiSAb may include additional linkers, such as a flexiblelinker interconnecting the variable heavy and light chains of an scFvand other linkers that connect other binding units to the BiSAb corestructure.

An exemplary, non-limiting example of a linker is a polypeptide chaincomprising at least 4 residues. Portions of such linkers may beflexible, hydrophilic and have little or no secondary structure of theirown (linker portions or flexible linker portions). Linkers of at least 4amino acids may be used to join domains and/or regions that arepositioned near to one another after the molecule has assembled. Longeror shorter linkers may also be used. Thus, linkers may be approximately1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, or approximately 50 residues in length. Whenmultiple linkers are used to interconnect portions of the molecule, thelinkers may be the same or different (e.g., the same or different lengthand/or amino acid sequence).

Linkers may be cleavable linkers, which contain at least one bond thatcan be selectively cleaved by a cleavage reagent. Cleavable linkers maybe used to facilitate removal of all or a portion of the linkersequence. Linkers may be engineered to contain protease cleavage sites,so that cleavage occurs in the middle of the linker or in at least oneend of the linker. For example, thrombin sites may be engineered at eachof the two flanking ends of a linker. Depending on the type of linkerused, cleavage may also be mediated by agents such as TCEP, TFA, andDTT. Linkers may be designed so that cleavage reagents remove allresidues from the linker from the cleavage product. Other exemplarynon-limiting linkers include prodrug linkers whose bonds can beselectively cleaved under in vivo conditions, for example, in thepresence of endogenous enzymes or other endogenous factors, or simply inaqueous fluids present in the body or in cells of the body. When BiSAbscontain more than one polypeptide linker, each of the linkers may bedifferent, or at least one of the linkers may be different from theothers. In some aspects a BiSAb comprises a cleavable linker. In aspecific aspect, the BiSAb comprises an scFv, wherein the scFv comprisesa cleavable linker between VH2 and VL2.

The linker(s) facilitate formation of the desired structure. Linkers maycomprise (Gly-Ser)_(n) residues, with some Glu or Lys residues dispersedthroughout to increase solubility. Alternatively or additionally linkersmay not comprise any Serine residues, such linkers may be preferablewhere the linker is subject to O-linked glycosylation. In some aspects,linkers may contain cysteine residues, for example, if dimerization oflinkers is used to bring the domains of the BiSAb into their properlyfolded configuration. In some aspects, the BiSAb comprises at least twopolypeptide linkers that join domains of the polypeptide. In otheraspects, the BiSAb comprises at least three polypeptide linkers. Inother aspects the BiSAb comprises four or more polypeptide linkers.

In some aspects, the polypeptide linker comprises a portion of an Fcmoiety. For example, in some aspects, the polypeptide linker cancomprise a portion of immunoglobulin hinge domain of an IgG1, IgG2,IgG3, and/or IgG4 antibody. In some aspects, the polypeptide linkercomprises a portion of a mutated immunoglobulin hinge domain of an IgG1,IgG2, IgG3 and/or IgG4. In some aspects, the polypeptide linkercomprises at least 5, 7, 8, or 15 amino acid residues of animmunoglobulin hinge region/domain of an IgG1, IgG2, IgG3, and/or IgG4antibody. In some aspects, the polypeptide linker comprises at least 5,7, 8, or 15 amino acid residues of a modified immunoglobulin hingeregion/domain of an IgG1, IgG2, IgG3, and/or IgG4 antibody.

The polypeptide linker may comprise all, or a portion of a hinge regionthat naturally comprises three cysteines. In certain aspects, theselected hinge region is truncated or otherwise altered or substitutedrelative to the complete and/or naturally-occurring hinge region suchthat only one or two of the cysteine residues remain. Similarly, incertain other aspects, the polypeptide linker may comprise a mutated orotherwise altered portion of a hinge region in which the number ofcysteine residues is reduced by amino acid substitution or deletion, forexample a mutated or otherwise altered hinge region containing zero, oneor two cysteine residues as described herein.

A mutated or otherwise altered hinge domain may thus be derived orconstructed from (or using) a wild-type immunoglobulin hinge domain thatcontains one or more cysteine residues. In certain aspects, a mutated orotherwise altered portion of a hinge region may contain zero or only onecysteine residue, wherein the mutated or otherwise altered hinge regionis or has been derived from a wild type immunoglobulin hinge region thatcontains, respectively, one or more or two or more cysteine residues. Inthe mutated or otherwise altered portion of a hinge region, the cysteineresidues of the wild-type immunoglobulin hinge region are preferablydeleted or substituted with amino acids that are incapable of forming adisulfide bond. In some aspects, a mutated or otherwise altered portionof a hinge region is or has been derived from a human IgG wild-typehinge region, which may include any of the four human IgG isotypesubclasses, IgG1, IgG2, IgG3 or IgG4.

In some aspects, the polypeptide linker comprises a portion of a hingeregion comprising the cysteine residue that forms a disulfide bond withan immunoglobulin light chain (EU residue 220). In some aspects, thepolypeptide linker comprises an altered portion of a hinge regioncomprising an amino acid substitution at EU residue C220. In someaspects, the polypeptide linker comprises the amino acid substitutionC220V.

In some aspects, the polypeptide linker comprises an amino acidsubstitution that prevents hinge-related spontaneous self-cleavage. Insome aspects, the polypeptide linker comprises an amino acidsubstitution at position at EU position D221. In some aspects, thepolypeptide linker comprises the amino acid substitution D221G. In someaspects, the polypeptide linker lacks the amino acid D221.

As discussed above, some embodiments include one or more polypeptidelinkers that comprise or consist of a gly-ser linker. As used herein,the term “gly-ser linker” refers to a peptide that consists of glycineand serine residues. An exemplary gly-ser linker comprises an amino acidsequence of the formula (Gly₄Ser)n, wherein n is a positive integer(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Some preferred and non-limitingexamples of a gly-ser linker includes (Gly₄Ser)₂, (SEQ ID NO:41) and(Gly₄Ser)₄, (SEQ ID NO:42) as well as (Gly₄Ser)₃ (SEQ ID NO:43). In yetother aspects, two or more gly-ser linkers are incorporated in series ina polypeptide linker. In some aspects, the polypeptide linker comprisesat least a portion of a hinge region (e.g., derived from an IgG1, IgG2,IgG3, or IgG4 molecule) and a series of gly-ser amino acid residues(e.g., a gly-ser linker such as (Gly₄Ser)n, where n is 2, 3, or 4).

In certain aspects, linkers (e.g., L1 and/or L2 and/or L3, etc.) includeboth a hinge portion and a linker portion, such as a linker portioncomprising a gly-ser linker. In other aspects, L1 and/or L2 include onlya hinge portion or only a linker portion, such as a gly-ser linker. Inother aspects, L1 and L2 include a gly-ser linker portion. In certainaspects, the gly-ser linker within a BiSAb is the same length, whereasin other aspects, the gly-ser linker portion within a BiSAb (e.g., L1and L2) are different lengths. When a BiSAb comprises an scFv, the heavyand light chains of the scFv may be connected to the BiSAb (e.g., BD1,Fab, Fc, etc.) by a flexible linker. This flexible linker generally doesnot include a hinge portion, but rather, is a gly-ser linker or otherflexible linker. The length and amino acid sequence of a flexible linkerinterconnecting domains of an scFv may be readily selected and optimized(e.g., (Gly₄Ser)n, (SEQ ID NO:48) where n is 2, 3, or 4 or more).

Regardless of the polypeptide linker used to interconnect variousbinding units and domains (e.g., between binding domains/units (e.g.,Fab-scFv), or binding domain/unit to Fc (e.g., scFv via L1 and L2), theBiSAb may optionally comprise additional polypeptide linkers. Thelengths and sequence of such additional polypeptide linkers areindependently selected. For example, the BiSAb may further comprise aflexible polypeptide linker interconnecting the variable heavy and lightchains of a scFv. This flexible polypeptide linker may comprise agly-ser linker. Generally, this linker does not include a hinge portion.

It is contemplated here that varying the length of the linkers flankingBD2 can impact on the orientation of the BD2 antigen binding site andspacing relative to the rest of the BiSAb molecule. For example, a shortN-terminal linker and long C-terminal linker may create an orientationwhere the binding site is conformed in one direction, while a longN-terminal and short C-terminal linker may impart an oppositeconformational orientation. Accordingly, linker length may be modulatedin order to orient the BD2 antigen binding site and have importantimpacts on creating or avoiding steric effects between BD1 and BD2and/or BD2 and other entities that bind the antibody molecule in the Fcor other domains.

5. Specific Configuration of BiSAbs

As discussed above, one aspect of the disclosure relates to a BiSAbstructural arrangement (platform) that comprises two heavy-light chainpairs (illustrated in FIGS. 1A-1F). In some embodiments of this aspect,the polypeptide sequence of the BiSAb chimeric heavy chain may comprisea polypeptide sequence comprising an antibody heavy chain variabledomain (VH1), a polypeptide sequence comprising an antibody heavy chainconstant domain 1 (CH1), a portion of the Fc domain, a polypeptidesequence comprising a first polypeptide linker (L1), a polypeptidesequence comprising a binding domain (BD2), a polypeptide sequencecomprising a second polypeptide linker (L2), and a polypeptide sequencecomprising the remainder of the Fc domain. In some aspects, the Fcdomain comprises a C_(H)2 domain and a C_(H)3 domain. Thus, certainembodiments provide a BiSAb chimeric heavy chain that may comprisepolypeptide sequences in the following orientation from N-terminus toC-terminus: VH1-C_(H)1-C_(H)2 (N-term)-L1-BD2-L2-C_(H)2 (C-term)-C_(H)3;VH1-C_(H)1-C_(H)2-L1-BD2-L2-C_(H)3; and VH1-C_(H)1-C_(H)2-C_(H)3(N-term)-L1-BD2-L2-C_(H)3 (C-term). The polypeptide sequence of theBiSAb light chain may comprise a light chain variable domain (VL1) and alight chain constant domain (CL). Thus, a BiSAb light chain may comprisepolypeptide sequence in the following orientation from N-terminus toC-terminus: VL1-CL. Note that VH1, VL1, and CL are used to denoteportions of “binding unit 1” (BD1) that binds a first epitope. BD2 isused to denote portions of “binding unit 2” that binds a second epitope.

In the aspects where the binding domain is an scFv, the BiSAb chimericheavy chain may comprise a polypeptide sequence comprising an antibodyheavy chain variable domain (VH1), a polypeptide sequence comprising anantibody heavy chain constant domain 1 (CH1), a polypeptide sequencecomprising a first polypeptide linker (L1), a polypeptide sequencecomprising an antibody light chain variable domain (VL2), a polypeptidesequence comprising a flexible linker, a polypeptide sequence comprisingan antibody heavy chain variable domain (VH2), a polypeptide sequencecomprising a second polypeptide linker (L2), and a polypeptide sequencecomprising an antibody Fc domain. Thus, the chimeric heavy chain of aBiSAb comprising an scFv as the BD2 may comprise a polypeptide sequencesin the following orientation from N-terminus to C-terminus:VH1-CH1-CH2(N-term)-L1-VL2-L3-VH2-L2-CH2(C-term)-CH3;VH1-CH1-CH2-L1-VL2-L3-VH2-L2-CH3;VH1-CH1-CH2-CH3(N-term)-L1-VL2-L3-VH2-L2-CH3(C-term);VH1-CH1-CH2(N-term)-L1-VH2-L3-VL2-L2-CH2(C-term)-CH3;VH1-CH1-CH2-L1-VH2-L3-VL2-L2-CH3; andVH1-CH1-CH2-CH3(N-term)-L1-VH2-L3-VL2-L2-CH3(C-term).

The chimeric heavy chain is a polypeptide chain comprising an amino acidsequence (e.g., the amino acid sequence of each of the polypeptidedomains). The chimeric heavy chain is a polypeptide chain comprising anamino acid sequence (e.g., the amino acid sequence of each of thepolypeptide domains). Note that VH1, VL1, and CL are used to denoteportions of binding unit 1, with VH1 and VL1 denoting that portion thatbinds the first epitope. VH2 and VL2 is used to denote portions ofbinding unit 2 that bind the second epitope. In certain aspects,additional scFv binding domains are present at the N-terminal and/orC-terminal ends of the polypeptides that make up the BiSAb core (whereinthe BiSAb core further comprises binding unit (BD) 3 and/or 4 and/or 5).In certain aspects, more than one scFv binding domains are presentwithin the BiSAb core. Each additional scFv comprises an antibody heavychain variable region denoted as VH3, VH4, VH5, and a correspondingantibody light chain variable region denoted as VL3, VL4, VL5.

6. Labels, Conjugates and Moieties

In certain features, drugs and other molecules may be targeted to BiSAbvia site-specific conjugation. For example, BiSAbs may comprise cysteineengineered domains (including cysteine(s) engineered into a binding unitand/or Fc domain), which result in free thiol groups for conjugationreactions. In certain aspects, a BiSAb is engineered to incorporatespecific conjugation sites. In some aspects, the present disclosureprovides an Fc variant BiSAb, wherein the Fc region comprises an aminoacid substitution at one or more of positions 239, 282, 289, 297, 312,324, 330, 335, 337, 339, 356, 359, 361, 383, 384, 398, 400, 440, 422,and 442, as numbered by the EU index. In some aspects, the Fc regioncomprises substitutions at one or more of the following groups ofpositions: a) 289 and 440; b) 330 and 440; c) 339 and 440; d) 359 and440; e) 289 and 359; f) 330 and 359; g) 339 and 359; h) 289 and 339; i)330 and 339; j) 289 and 330; k) 339 and 442; l) 289, 339, and 442; m)289, 330, and 339; n) 330, 339, and 442; and o) 289, 330, and 442. Inother aspects, the present disclosure provides a BiSAb, wherein the CH1domain of the Fab arm comprises a substitution at one or more ofpositions 131, 132, 134, 135, 136 and 139, as numbered by the EU index.In one aspect the substitution comprises a substitution to an amino acidchosen from cysteine, lysine, tyrosine, histidine, selenocysteine, andselenomethionine. In a specific aspect, the substitution is a cysteine.Methods for generating stable cysteine engineered antibodies aredescribed in U.S. Pat. No. 7,855,275, U.S. 20110033378 andUS20120213705, the contents of which are incorporated herein byreference in their entirety.

7. Exemplary Targets

While the aspects and embodiments relating to the various DuetMab andBiSAb platform(s) described herein can be generated to bind to anydesired target or targets, the BiSAbs disclosed herein preferably targetspecific pairs of target molecules (e.g., binding unit 1 binds one ofthe targets and binding unit 2 binds the other target). As discussedabove and as exemplified in the illustrative Examples below, theantibodies, DuetMabs and BiSAbs disclosed herein are targeted to amolecule that modulates an immune response in a recipient subject, or inimmune cells in culture. In some embodiments the binding domain exhibitsspecific binding activity for a target selected from the groupconsisting of CTLA-4, PD-1, PD-L1, OX40, and TIM3. The DuetMabs andBiSAbs can comprise a combination of different binding domains invarious orders and orientations, where the domains have binding affinityfor, or bind specifically to the targets disclosed herein. For examplethe DuetMabs and BiSAbs disclosed herein may comprise a combination ofbinding domains that allow for bispecific binding to targets including;CTLA-4 and PD-1; CTLA-4 and PD-L1; and CTLA-4; CTLA-4 and TIM3; PD-1 andPD-L1; PD-L1 and OX40; PD-1 and TIM3; PD-L1 and TIM3. DuetMabs andBiSAbs that include binding domains that bind particular targetcombinations are illustrated in the Examples and include thenon-limiting combinations of PD-1/CTLA-4; PD-L1/CTLA-4; PD-1/TIM3; andPD-L1/OX40. In certain embodiments, the BiSAbs have enhanced bindingproperties relative to the binding properties of the combined individualmonospecific binding proteins that are used to generate the BiSAbs.

In certain aspects, a DuetMab or BiSAb of the disclosure binds twodifferent epitopes on the same target (e.g., binding unit 1 binds afirst epitope on a target and binding unit 2 binds a second epitope onthe same target).

In some aspects, the multimeric nature of the DuetMabs or BiSAbs of thedisclosure confers the ability to target labels or therapeutics to aspecific cell type or molecular target. For example, one functionaldomain in a DuetMab or BiSAb may bind to a target at the surface of acell, while another functional domain in the same DuetMab or BiSAb bindsto a hapten or labeling agent useful for detection. Similarly, onefunctional domain may bind to a cellular target while a secondfunctional domain binds to a toxin. Because both binding reactions aremediated through a single molecule, the toxin may be placed in theproximity of the cellular target, where it affects a cytotoxic function.

B. Nucleic Acid Molecules Encoding BiSAbs

The present disclosure provides nucleic acid molecules that encodeBiSAbs. One aspect of the disclosure provides nucleic acid moleculesencoding any of the BiSAbs of the disclosure. A nucleic acid moleculemay encode a heavy chain and/or light chain of any of the BiSAbmolecules that are disclosed herein, as well as any of the individualbinding domains (e.g., scFvs) that are disclosed herein. One of skill inthe art will appreciate that such polynucleotide molecules may vary innucleotide sequence given nucleic acid codon degeneracy as well as codonfrequency for particular organisms, as is generally known in the art.

C. Vectors and Host Cells for Producing BiSAbs and SubsequentPurification

The disclosure relates to methods for producing BiSAbs. In certainaspects, recombinant nucleic acid molecules that encode all or a portionof the BiSAbs disclosed herein may be operably linked to one or moreregulatory nucleotide sequences in an expression construct. The nucleicacid sequences encoding the BiSAb light and chimeric heavy chains can becloned in the same expression vector in any orientation (e.g., lightchain in front of the heavy chain or vice versa) or can be cloned in twodifferent vectors. If expression is carried out using one vector, thetwo coding genes can have their own genetic elements (e.g., promoter,RBS, leader, stop, polyA, etc) or they can be cloned with one single setof genetic elements, but connected with a cistron element. Regulatorynucleotide sequences will generally be appropriate for a host cell usedfor expression. Numerous types of appropriate expression vectors andsuitable regulatory sequences are known in the art for a variety of hostcells. Typically, said one or more regulatory nucleotide sequences mayinclude, but are not limited to, promoter sequences, leader or signalsequences, ribosomal binding sites, transcriptional start andtermination sequences, translational start and termination sequences,and enhancer or activator sequences. Constitutive or inducible promotersas known in the art are contemplated by the disclosure. The promotersmay be either naturally occurring promoters, or hybrid promoters thatcombine elements of more than one promoter. An expression construct maybe present in a cell on an episome, such as a plasmid, or the expressionconstruct may be inserted in a chromosome.

In certain aspects, the expression vector contains a selectable markergene to allow the selection of transformed host cells. Selectable markergenes are well known in the art and will vary with the host cell used.In certain aspects, this disclosure relates to an expression vectorcomprising a nucleotide sequence encoding a polypeptide and operablylinked to at least one regulatory sequence. Regulatory sequences areart-recognized and are selected to direct expression of the encodedpolypeptide. Accordingly, the term regulatory sequence includespromoters, enhancers, and other expression control elements. Exemplary,non-limiting regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). It should be understood that the design of the expressionvector may depend on such factors as the choice of the host cell to betransformed and/or the type of protein desired to be expressed.Moreover, the copy number of the particular vector, the ability tocontrol that copy number and the expression of any other protein encodedby the vector, such as antibiotic markers, should also be considered.

The disclosure further pertains to methods of producing a BiSAb of thedisclosure. For example, a host cell transfected with one or more thanone expression vector encoding a BiSAb (e.g., a single vector encodingthe chimeric heavy and the light chain or two vectors, one encoding thechimeric heavy chain and one encoding the light chain) can be culturedunder appropriate conditions to allow expression of the polypeptide tooccur. The BiSAb may be secreted and isolated from a mixture of cellsand medium containing the polypeptide. Alternatively, the BiSAb may beretained in the cytoplasm or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. BiSAbs can be isolated from cell culture medium,host cells, or both using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification. In certain aspects, the BiSAb is made as a fusion proteincontaining a domain which facilitates its purification.

A recombinant nucleic acid can be produced by ligating the cloned gene,or a portion thereof, into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian),or both. Expression vehicles for production of a recombinant polypeptideinclude plasmids and other vectors. For instance, suitable vectorsinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli. Incertain aspects, mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Ban virus (pHEBo, pREP-derived and p205) can be usedfor transient expression of proteins in eukaryotic cells. The variousmethods employed in the preparation of the plasmids and in thetransformation of host organisms are known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press, 1989) Chapters 16 and 17. In someinstances, it may be desirable to express the recombinant polypeptide bythe use of a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUW1), andpBlueBac-derived vectors (such as the β-gal containing pBlueBac III).

Once a molecule has been produced, it may be purified by any methodknown in the art for purification of a protein, an immunoglobulinmolecule or other multimeric molecules using techniques such as, forexample, chromatography (e.g., ion exchange, affinity, particularly byaffinity for the specific antigens Protein A or Protein G, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins. Further,the molecules disclosed herein may be fused to heterologous polypeptidesequences (e.g., affinity tags) as are routinely employed to facilitatepurification.

Regardless of how a BiSAb is generated and purified, binding assays, forexample, dual ELISA assays, may be performed (before and/or afterpurification) to confirm functional binding activity of the BiSAb. Suchbinding assays are generally known in the art.

D. Pharmaceutical Formulations

In certain aspects, the disclosure provides pharmaceutical compositions.Such pharmaceutical compositions may be compositions comprising anucleic acid molecule that encodes a BiSAb. Such pharmaceuticalcompositions may also be compositions comprising a DuetMabs, a BiSAb, acombination of DuetMabs, or a combination of BiSAbs, and apharmaceutically acceptable excipient. In certain aspects, thepharmaceutical compositions of the disclosure are used as a medicament.

E. Uses

As discussed herein the DuetMabs and BiSAbs may be used to bind targetsassociated with cancer and cell proliferative diseases or disorders thatmay be responsive to an immunotherapy, for example, by inhibiting animmunosuppressive activity and/or by inducing an immune response that isassociated with the target molecule(s). For example, aberrant signallingand/or inhibited immune response may contribute to unwanted cellproliferation and cancer. Accordingly, DuetMabs. BiSAbs and theantibodies disclosed herein may be used to treat unwanted cellproliferation and/or cancer associated with an inhibited, reduced, orinsufficient immune response targeted against the cancer. In particular,the tumor growth curve of a tumor and/or the volume of a tumor may bereduced by administration of a DuetMab of BiSAb that induces and/orstimulates an immune response in a subject, such as, for example a humanpatient suffering from a cancer.

Thus, the disclosure also relates to various methods that compriseadministration of the binding proteins disclosed herein to a subject inneed thereof. In one aspect, the disclosure relates to a method forinducing an immune response in a subject having, or at risk ofdeveloping, a cancer comprising administration of a binding proteindisclosed herein to the subject. In some embodiments, the methodactivates an immune response against the cancer in the subject. In someembodiments, the method enhances an immune response against the cancerin the subject. In some embodiments, the method activates an immuneresponse pathway that is inhibited in the subject, wherein theactivation increases an immune response that targets the cancer in thesubject. In some embodiments the method enhances an immune responsepathway that targets the cancer in the subject.

In another aspect, the disclosure relates to a method for treatingcancer in a subject in need thereof comprising administering a bindingprotein disclosed herein to the subject. In one embodiment the method oftreating cancer comprises stopping or slowing the growth of the cancerin the subject. In one embodiment the method of treating cancercomprises stopping or slowing the metastasis of the cancer to otherareas in the subject. In one embodiment the method of treating cancercomprises killing cancer cells in the subject. In one embodiment themethod of treating cancer comprises halting the proliferation and/or thespread of cancer cells in the subject.

In various embodiments of the above aspects, the methods relate totreating a subject for a tumor disease and/or a cancer disease. Inembodiments the cancer is selected from the group of cancers that aresusceptible to an immune response induced in the subject. In someembodiments, the cancer is one or more of an ovarian cancer, breastcancer, colorectal cancer, prostate cancer, cervical cancer, uterinecancer, testicular cancer, bladder cancer, head and neck cancer,melanoma, pancreatic cancer, renal cell carcinoma, or lung cancer. Insome embodiments the cancer is selected from digestive orgastro-intestinal cancers (e.g., anal cancer; bile duct cancer;extrahepatic bile duct cancer; appendix cancer; carcinoid tumor,gastrointestinal cancer; colon cancer; colorectal cancer includingchildhood colorectal cancer; esophageal cancer including childhoodesophageal cancer; gallbladder cancer; gastric (stomach) cancerincluding childhood gastric cancer; hepatocellular cancer (e.g.,hepatocellular carcinoma) including adult (primary) hepatocellularcancer and childhood hepatocellular cancer; pancreatic cancer includingchildhood pancreatic cancer; sarcoma, rhabdomyo sarcoma; islet cellpancreatic cancer; rectal cancer; and small intestine cancer); lungcancer (e.g., non-small cell lung cancer (NSCLC) and small cell lungcancer (SCLC)); head and neck cancer (e.g., lip and oral cavity cancer;oral cancer including childhood oral cancer; hypopharyngeal cancer;laryngeal cancer including childhood laryngeal cancer; metastaticsquamous neck cancer with occult primary; mouth cancer; nasal cavity andparanasal sinus cancer; nasopharyngeal cancer including childhoodnasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer;pharyngeal cancer; salivary gland cancer including childhood salivarygland cancer; throat cancer; and thyroid cancer); ovarian and breastcancer.

In the above methods, the amount of binding protein that is administeredto the subject is effective to induce an immune response, increase animmune response, stop or slow the growth of cancer, stop or slow themetastasis of cancer, kill cancer cells, and/or slow or stop theproliferation and/or spread of cancer cells in the subject.

In embodiments of the above methods, the binding protein comprises aDuetmAb or BiSAb as disclosed herein. In some embodiments of the abovemethods, the binding protein comprises an antibody, or anantigen-binding fragment thereof, as disclosed herein.

As used herein, the term “subject” is intended to include human andnon-human animals, particularly mammals. Examples of subjects includehuman subjects for example a human patient having a disorder, e.g., adisorder described herein, such as cancer, or a normal subject. A“non-human animal” includes all vertebrates, e.g., non-mammals (such aschickens, amphibians, reptiles) and mammals, such as non-human primates,domesticated and/or agriculturally useful animals (such as sheep, dogs,cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters,guinea pigs, etc.). In particular embodiments, the subject is a humanpatient.

“Treatment” or “treat” refers to both therapeutic treatment andprophylactic or preventative measures. Those subjects in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in which the disorder is to be prevented.When used with reference to a disease or a subject in need of treatmentthe terms accordingly include, but are not limited to, halting orslowing of disease progression, remission of disease, prophylaxis ofsymptoms, reduction in disease and/or symptom severity, or reduction indisease length as compared to an untreated subject. In embodiments, themethods of treatment can abate one or more clinical indications of theparticular disease being treated.

The Examples that follow are provided to illustrate particular aspectsand embodiments of the disclosure provided above and should not beinterpreted as limiting to the scope of the description or to theappended claimed subject matter.

EXAMPLES Materials and Methods

Immune Response Modulation Assay

A cytomegalovirus (CMV) antigen recall assay was used to evaluate thepotential immune response induced by certain of the immunotherapeuticmolecules described herein. Reagents for the assay include:

-   -   CMV reactive frozen peripheral blood mononuclear cells (PBMC);    -   AIM V® Medium (Life Technologies, cat#12055-091);    -   phosphate buffered saline (PBS, Life technologies,        cat#20012-043);    -   PepTivator®, CMVpp65 peptide pool, (Miltenyi Biotec,        cat#130-093-438, 50 μg/ml);    -   Ovalbumin, (Thermo scientific cat#77120, 1 mg/ml);    -   Costar, 96 well plate non-TC treated (Corning, cat#3788); and    -   an immunotherapeutic molecule.

General Assay Protocol:

The day before the assay was performed frozen PBMCs were thawed in warmAIM V medium. The cells were washed twice in Costar 96 round well plate.The concentration of cells was adjusted to a concentration of 1×10⁶cells/mL.

Aliquots (100 μL) of cells were dispensed in individual wells, leavingthe outside columns and rows of the plate empty. The cells were allowedto rest overnight.

The following day, 100 μL of AIMV medium containing 2× PepTivator CMVpeptide pool (0.1 μg/ml-0.05 μg/ml final) and 2× immunotherapeuticmolecule were added to the wells.

After 72 hours, 25 μL of supernatant from each well was transferred to apre-blocked and washed MSD plates (anti-human IFN gamma). After additionof standards, plates were incubated for 2 hours at room temperature.After the incubation period, the MSD plates were washed three times.Following the washing, 25 μL of SULFO-TAG detection antibody was addedand allowed to react for 1 hour at room temperature. The plates werewashed again and 150 μL of 2λ MSD read buffer was added before readingswere taken.

Staphylococcal Enterotoxin A/B (SEA/SEB) Assay Protocol

Reagents used in either the SEB or SEA assay protocol to determine theeffect of the DuetMabs or BiSAbs on IL-2 immune response include:

-   -   Leukocyte cones (NHSBT code NC24; from Addenbrookes Hospital);    -   50 ml Falcon tubes (BD 352070);    -   Ficoll-Paque PLUS (GE Healthcare 17-1440-02);    -   Anti-CD3 (clone OKT3; 1 mg/ml; eBioscience; cat no:16-0037-85);    -   Ammonium chloride solution (Stemcell Technologies 07850);    -   Staphylococcal enterotoxin A (SEA; Sigma, S-9399) or        Staphylococcal enterotoxin B (SEB; Sigma, S-4881) stock        solutions at 1 mg/ml stored at −20° C.;    -   Culture media (all from Life Technologies): RPMI1640 with        glutamax (61870) supplemented with 10% v/v heat inactivated FCS        (90005M) and 100 U/ml penicillin+100 ug/ml streptomycin        (15140-122);    -   V-bottomed plate (Greiner BioOne 651201);    -   96-well flat-bottom plates (Corning Costar 7107).

Reagents for the IL-2 DELFIA ELISA include:

-   -   FLUONUNC Maxisorp ELISA plates (Nunc 437958);    -   Europium-labelled streptavidin, SA-Eu (Perkin-Elmer 1244-360);    -   DELFIA® assay buffer (Perkin-Elmer, #4002-0010);    -   DELFIA® enhancement solution (Perkin-Elmer 4001-0010); at RT        prior to use;    -   Assay diluent: DELFIA wash buffer (0.05% Tween-20, 20 mM Tris,        150 mM NaCl; pH 7.2-7.4) supplemented with 0.1% BSA, sterile        filtered;    -   Milk powder (Marvel; Premier Foods);    -   Sample Diluent (RPMI1640+10% FCS+1% Penicillin/Streptomycin as        above);    -   PBS (ThermoFisher 14190235);    -   PBS-Tween (0.01% Tween-20 in PBS);    -   Human IL-2 ELISA kit (Duoset DY202, R&D Systems);    -   Biotek plate washer (EL406) with automated plate loader        (Biostack).

General Assay Protocol

PBMCs were isolated from human blood leukocyte cones (NHS Blood andTransplant Service code NC24) using density gradient centrifugation(Ficoll-Paque PLUS; GE Healthcare), then red blood cells were lysed inammounium chloride solution (Stemcell Technologies). Anti-human CD3(clone OKT3 at 0.5 ug/ml in PBS; eBioscience) was coated inflat-bottomed 96 well plates (Corning Costar 7107) for 2 hrs at 37° C.Then, 0.2×10⁶ cells were added, per well, of the PBMC in culture media(RPMI1640-Glutamax supplemented with 10% v/v heat inactivated bovineserum and 100 U/100 ug per ml Steptomycin/Penicillin (respectively)(Life Technologies). PBMC were further stimulated by addition ofStaphylococcal Enterotoxin A or B (SEB; Sigma Aldrich) within a range of0.0088-0.1 ug/mL, and candidate DuetMabs or BiSAbs were added to thefinal tested concentrations. Following 3 days culture at 37° C. and 5%CO₂ supernatants were removed from cells and IL-2 secretion determinedusing commercial ELISA according to manufacturer's instructions (R&DSystems Duoset product code DY202). See FIG. 90.

Mixed Leukocyte Reaction (MLR) Assay Protocol (Fresh Blood)

The MLR cell-based assay was also used to provide in vitro correlationof T cell function in response to the DuetMabs and BiSAbs disclosedherein. Reagents used in performing the MLR assay from fresh bloodsamples include:

-   -   8 mL CPT Heparin tubes;    -   AIM-V Medium (serum free) Gibco #12055-091, no additives;    -   50 ml conical tubes;    -   2 ml cryopreservation vials;    -   ACK lysing Buffer (Gibco # A10492-01);    -   96 well tissue culture treated U-bottom plates BD falcon #3077;    -   PHA (Roche) 1 mg/ml (10 ug/mL final concentration), as a        positive control;

General Assay Protocol

PBMCs were prepared from blood samples drawn into CPT Heparin tubes. Thetubes are centrifuged for 20 min at 2700 rpm without the brake at 25° C.The top layer of serum is aspirated. The remaining material was gentlypipetted, and everything above CPT tube plug was collected and placedinto 50 ml conical tubes. To the cells was added AIM-V medium to washthe cells (3 times at 1500 rpm, with the brake on, at 25° C. for 5minutes). Any remaining red blood cells were lysed using red blood celllysing buffer (e.g., about 5 min. with about 3 ml buffer). The remainingcells were washed twice with AIM-V medium (1500 rpm, break on, at 25° C.for 5 minutes). If needed, the pellets were consolidated into a singletube and resuspended in AIM-V medium, and a cell count was made.

To perform the MLR assay, the cells were plated into 96 well plates at200,000 cells per donor per well in AIM-V medium, 50 ul per donor (totalof 400,000/100 ul). The candidate molecules were added (4×) 50 ul perwell diluted in Serum Free AIM-V Medium. After 72 hrs., the plates wereimaged and 30 ul of supernatant was removed for human TH1/TH2 (MSD)cytokine assay.

Human TH1/TH2 MSD 10-Plex Protocol

This assay was used to determine amounts of cytokines present in culturesupernatants in response to the administration of the DuetMabs andBiSAbs disclosed herein. To perform the assay, blocker agent wasprepared by dissolving 200 mg of blocker B into 20 ml PBS per plate. 150ul dissolved blocker was added to each well. The plate was sealed andshaken for 2 hrs at room temperature or overnight at 4° C. The wellswere washed 3× with PBST buffer. A calibrator was prepared by dilutingfrozen calibrator blend 10 ul into 1 ml of diluent, and was furtherserially diluted 4 fold. To separate wells was added 25 ul of calibrator(standard) and 25 ul samples. The wells were incubated for 2 hrs, atroom temperature with shaking. Following incubation, the wells werewashed 3× with PBST.

Detection antibody is prepared and diluted to the necessaryconcentration, and was added to each well. Following a 2 hr. incubationat room temperature with shaking, the wells were washed (3×) in PBST.Prior to reading on the MSD machine, read buffer was added to each well.

Tumor Specific Killing Assay Protocol

The human CD8+ T cell line (JR6C12) with reactivity against humangp100₂₀₉₋₂₁₇ peptide was kindly provided by Dr. Steven Rosenberg(National Cancer Institute, Bethesda, Md.). JR6C12 cells wereco-cultured with a CFSE (CellTrace CFSE proliferation kit, ThermoFisher)labeled human melanoma line (Mel624) for 18 hours at 37 C at a 1:1 ratio(20,000 JR6C12+20,000 Mel624) in a 96 well flat bottom plate. Thecandidate molecules were added at time 0 of the co-culture at aconcentration of 69 nM. After 18 hrs, wells were visualized by brightfield microscopy. Supernatants were collected for MSD analysis andadherent cells were trypsinized and washed (2×) in PBS prior toviability staining (Zombie UV Fixable Viability kit, Biolegend).Viability dye uptake of CFSE labeled cells was assessed by flowcytometry on a LSRFortessa (BD).

Example 1. Identification of Candidate Fc Locations for Binding DomainAttachment

Using the open-source software PyMOL molecular visualization system,antibody structure was investigated in the CH2 and CH3 regions as wellas at or near the CH2-CH3 interface in order to identify candidateregions, such as exposed surface loops, for binding domain attachment.Such regions would accommodate for insertion of a second binding domain(e.g., an scFv) without compromising the structural integrity orstability of the IgG or the second binding domain itself. From theanalysis, three regions were identified (represented as spheres in FIGS.1A-1C). FIGS. 1D, 1E, and 1F depict embodiments of the binding proteins,showing attachment of a second binding domain (with an scFv for purposesof illustration) in each of the loops identified in FIGS. 1A, 1B, and1C, respectively.

FIG. 2A provides a more detailed schematic diagram of the amino acidsequence of one of the identified representative loops identified in theCH2 region near the CH2-CH3 interface and comprising the sequence ISRTP(SEQ ID NO:39). A binding domain may be inserted within this amino acidsequence to generate any number of representative constructs such as,for example, inserting scFv domains as illustrated in the Examples(e.g., I-scFv-SRTP, IS-scFv-RTP, ISR-scFv-TP, or ISRT-scFv-P scFV-ISRTP,and ISRTP-scFV, where the “-scFv-” identifies the point in the nativeloop sequence to which the binding domain may be associated. FIG. 2B isa similar schematic diagram that is representative of the loopidentified in the CH2-CH3 interface and comprising the amino acidsequence AKGQP (SEQ ID NO:40). Representative constructs describedherein can include a binding domain (such as, for example, a scFvdomain) attached to this loop sequence as described herein, includingA-scFv-KGQP, AK-scFv-GQP, AKG-scFv-QP, AKGQ-scFv-P, scFV-AKGQ,AKGQ-scFV, where the “-scFv-” identifies the point in the native loopsequence to which the binding domain may be associated. FIG. 2C providesa schematic diagram of the representative loop identified downstream ofthe CH2-CH3 interface, within the CH3 region and comprising the aminoacid sequence SNG. The representative constructs for this loop sequence,discussed in terms of the illustrative embodiments for the other twoloop regions above, include scFV-SNG, S-scFv-NG SN-scFv-G, and SNG-scFV.

Example 2. Generation and Characterization of a Series of ParentalAntibodies and Bispecific Binding Proteins Including Combination ofBinding Units

A series of monoclonal antibodies were developed and characterized.Using combinations of antigen-binding sequences (e.g., CDRs, HCv, LCv,HC, LC) derived from these “parental” antibodies a series of bispecificbinding proteins were generated, and shown to have bispecific bindingactivity for the combined target antigens. The bispecific bindingproteins were designed to have the particular structural platform motifwhich is disclosed herein (i.e., “BiS5”).

Parental antibody sequences are described in the following Tables:

TABLE 2a Parental antibody sequences Description/ Target Sequence PD-1PD-1 LC QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQAPRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) PD-1 LCvQIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQAPRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 49) PD-1 HCEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 50) PD-1 HCvEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSS (SEQ ID NO: 51) CTLA-4 CTLA-4 HCvQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 52) CTLA-4 HCGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH (SEQ ID NO: 53) CTLA-4 LCvDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO: 54)CTLA-4 LC DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55)PD-L1 PD-L1 EVQLVESGGGLVQPGRSLRLSCTASGYTFPDYYMNWVRQAPGKGLEWVGDI (AMP714)DPNYGGTTYNASVKGRFTISVDRSKSIAYLQMSSLKTEDTAVYYCARGALTD HCvWGQGTMVTVSS (SEQ ID NO: 56) PD-L1QIQLTQSPSILSASVGDRVTITCRASSSVSYIYWFQQKPGKAPKPLIYATFNLAS (AMP714)GVPSRFSGSGSGTSYTLTISSLQPEDFATYYCQQWSNNPLTFGQGTKVEIKRTV LCAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 57)PD-L1 QIQLTQSPSILSASVGDRVTITCRASSSVSYIYWFQQKPGKAPKPLIYATFNLAS (AMP714)GVPSRFSGSGSGTSYTLTISSLQPEDFATYYCQQWSNNPLTFGQGTKVEIK LCv (SEQ ID NO: 58)PD-L1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANI (MEDI4736)KQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGW HCvFGELAFDYWGQGTLVTVSS (SEQ ID NO: 59) PD-L1EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASS (MEDI4736)RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK LCRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 33)PD-L1 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASS (MEDI4736)RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK LCv(SEQ ID NO: 60) TIM3 TIM3 (WT)QTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSD #62 LCRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 61) TIM3 (WT)QTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSD #62 LCvRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGGGTKLTVL (SEQ ID NO: 62) TIM3 (WT)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS #62 HCGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 63) TIM3 (WT)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS #62 HCvGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSS (SEQ ID NO: 64) TIM3SYVLTQPPSVSVAPGKTARITCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSD (germlined)RPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDRRSDHWLFGGGTKL #62 LCTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 65) TIM3SYVLTQPPSVSVAPGKTARITCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSD (germlined)RPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDRRSDHWLFGGGTKL #62 LCvTVL (SEQ ID NO: 66) TIM3EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS (germlined)GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGT #62 HCYYGNYFEYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 67) TIM3EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS (germlined)GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGT #62 HCvYYGNYFEYWGQGTLVTVSS (SEQ ID NO: 68) OX40 OX40 HCvQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS (SEQ ID NO: 69) OX40 LCvDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGS GSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK (SEQ ID NO: 70)OX40 HC QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71) OX40 LCDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 72)

TABLE 2b Antigen sequences Description/ Target Sequence PD-1MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNAT humanFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 73) PD-L1MRIFAVFIFMTYWHLLNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIW humanTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 74) CTLA-4MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRG humanIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 75) TIM3MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVC humanWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP (SEQ ID NO: 76) OX40MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNG humanMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 78)

Example 2(a) PD-1/CTLA-4 Bispecific Binding Proteins

Without being bound by theory, there is a strong clinical andpreclinical rationale for the combination of PD-1 and CTLA-4 blockade.Thus, it would be desirable to maximize the risk/benefit ratio of PD-1and CTLA-4 combination (FIG. 4).

The following bispecific binding proteins that bind PD-1 and CTLA-4 werecreated using the parental sequences identified above in Table 2.Proteins identified as Bis2, Bis3, and Bis5 were generated with thesequences identified below and were assessed for concurrent antigenbinding activity using the Octet binding assay as discussed below.

TABLE 3 BIS constructs for PD-1/CTLA-4 Description Sequence Bis2DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIY PD-1/CTLA-4AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFG HCCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1) Bis2QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1/CTLA-4PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) Bis3EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1/CTLA-4WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY HCYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3) Bis3QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1/CTLA-4PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4) Bis5EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1/CTLA-4WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY HCYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK (SEQ ID NO: 5)Bis5 QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1/CTLA-4PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)

Octet Binding Assay (BiS2, BiS3, and BiS5)

To evaluate binding of the bispecific binding molecules disclosedherein, an Octet QK equipped with Ni-NTA biosensor tips and 10× kineticsbuffer were used (ForteBio, Menlo Park, Calif.). For this particularseries of bispecific binding proteins, His-tagged PD-L1-Fc, his-taggedPD-1-Fc and CTLA-4-Fc (human recombinant proteins) were purchased fromR&D Systems (Minneapolis, Minn.). All binding assays were performed at25° C.

Sample plates were agitated at 1000 rpm prior to analysis. The Ni-NTAbiosensor tips were pre-wetted for 5 min. in 1× kinetic buffer. The 1×kinetic buffer also served as the running buffer for baselinedetermination and as the dilution buffer for antigens and bispecificantibodies. Ni-NTA biosensor tips were dipped into 100 nM his-taggedPD-L1-Fc (see, (b), below) or his-tagged PD-1-Fc for antigen capture forabout 1 min. The antigen-coated biosensor tips were each dipped into 10μg/ml bispecific antibodies for ˜5 minutes and then moved into a columnof wells containing 100 nM CTLA-4-Fc antigen for 2 minutes. The bindingresults are shown in FIG. 3.

A bispecific binding protein in DuetMab format that binds PD-1 andCTLA-4 was created using the parental sequences identified above inTable 2. The PD-1/CTLA-4 DuetMab was generated with the sequences inTable 4 below and was assessed as discussed below, including incomparison with PD-1/CTLA-4 Bis5.

TABLE 4 DuetMab constructs for PD-1/CTLA-4 Description Sequence DuetMabQIVLTQSPATLSLSPGERATLSC

QQKPGQA PD-1 (LO115) PRLLIY

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC

LC

FTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE Amino acidAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7) DuetMabCAGATCGTGCTGACCCAGTCCCCTGCCACCCTGTCCCTGAGCCCTGG PD-1(LO115)CGAGAGAGCCACCCTGAGCTGCTCCGCCTCCTCCAAGCACACCAAC LCCTGTACTGGTCCCGGCACATGTACTGGTATCAGCAGAAGCCCGGCC Nucleic acidAGGCCCCTCGGCTGCTGATCTACCTGACCTCTAACCGGGCCACCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCCTCCAACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 8) DuetMab EVQLVESGGGLVQPGGSLRLSCAAS

WVRQAPGKGLE PD-1 (LO115) WVA

RFTISRDNAKNSLYLQMNSLRAEDTAVYY HC CAR

WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGT Amino acidAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) DuetMabGAGGTGCAGCTGGTGGAATCCGGCGGAGGACTGGTGCAGCCTGGC PD-1 (LO115)GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACATTCTCCGA HCCTACGGCATGCACTGGGTCCGACAGGCCCCTGGAAAGGGCCTGGAA Nucleic acidTGGGTGGCCTACATCTCCTCCGGCTCCTACACCATCTACTCCGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACAGCCGTGTACTACTGTGCCAGACGGGCCCCTAACTCCTTCTACGAGTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCTAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCGAGCAGCAAAAGCACCTCTGGTGGCACCGCAGCACTGGGTTGTCTGGTGAAAGATTATTTTCCGGAACCGGTTACCGTTTCTTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTTCTGCAGAGCAGCGGTCTGTATAGCCTGTCTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAATGTGAATCATAAACCGAGCAATACAAAAGTTGATAAACGCGTTGAACCGAAAAGCTGTGACAAAACTCACACGTGCCCACCGTGCCCAGCACCTGAGTTCGGAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCAGATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTGCACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAGCTGCGCGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 10) DuetMab DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY CTLA-4

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

FTFGP LC GTKVEIKGQPKAAPSVTLFPPCSEELQANKATLVCLISDFYPGAVTVAW Amino acidKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS (SEQ ID NO: 4) DuetMabGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG CTLA-4AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAACAGC LCTATTTAGATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCC Nucleic acidTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATTACAGTACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAAGGTCAGCCCAAGGCGGCCCCCTCGGTCACTCTGTTCCCGCCCTGCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAGTGTCA (SEQ ID NO: 11) DuetMabQVQLVESGGGVVQPGRSLRLSCAAS

WVRQAPGKGLE CTLA-4 WVA

RFTISRDNSKNTLYLQMNSLRAEDTAV HC YYCAR

WGQGTTVTVSSASTKGPSVCPLAPSSK Amino acidSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12) DuetMabCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA CTLA-4GGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGC HCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGT Nucleic acidGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCCGAGGGGAGCTACCCTTTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCGTCGACCAAAGGTCCGAGCGTGTGCCCGCTGGCACCGAGCAGCAAAAGCACCTCTGGTGGCACCGCAGCACTGGGTTGTCTGGTGAAAGATTATTTTCCGGAACCGGTTACCGTTTCTTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTCCTGCAGAGCAGCGGTCTGTATAGCCTGTCTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAATGTGAATCATAAACCGAGCAATACCAAAGTTGATAAACGCGTTGAACCGAAAAGCGTGGACAAAACTCACACGTGCCCACCGTGCCCAGCACCTGAGTTCGAGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCagCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATGCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCTTAAGCCTGTCTCCGGGTAAA (SEQ ID NO: 13)

Octet Binding Assay (DuetMab)

Concurrent binding studies to two distinctive antigens were performed byOctet analysis. Biotinylated human PD-1 was loaded on Streptavidinsensors followed by sequential interactions first with DuetMabPD-1/CTLA-4 and then with soluble CTLA-4 antigen. Streptavidin (SA)biosensors (ForteBio) were used to capture biotinylated human PD-1 at 5μg/ml in PBS pH 7.2, 3 mg/ml BSA, 0.05% (v/v) Tween 20 (assay buffer).Following a washing step the loaded biosensors were subjected forsuccessive association and dissociation interactions first with samplewells carrying the DuetMab PD-1/CTLA-4 bispecific antibody at 133 nM andthen with wells carrying human CTLA-4 antigen at 200 nM. The bindingresults are shown in FIG. 5.

Intrinsic kinetics of the PD-1/CTLA-4 DuetMab bispecific antibody wasalso assessed via BiaCore. Binding experiments were carried out using aBIAcore T200 instrument (BIAcore). To capture the antibody, mouseanti-huIgG-Fab was immobilized on a CM5 chip to a target response of2000 RU. 100 nM of the DuetMab or mAbs were flowed at 20 μL/min for 5min to achieve approximately 100 response units of captured antibody.Antigen were then injected serially at a flow rate of 50 μl/min for 5min. Kinetic parameters (k_(on) and k_(off)) and dissociation constant(KD) were calculated from a non-linear fit using BIAevaluation 4.1software. The binding results are shown in Table 5.

TABLE 5 BiaCore data for PD-1/CTLA-4 kon koff K_(D) Capture Surface PD1CTLA4 (×E + 5 M⁻¹s⁻¹) (×E − 4 s⁻¹) (nM) Chi² Anti-PD-1 mu-anti-huIgG hu3.02 2.37 0.79 0.08 (LO115) IgG mu-anti-huIgG cyno 3.46 2.38 0.69 0.06mu-anti-huIgG mu 1.49 665 447 0.13 Anti-CTLA-4 mu-anti-huIgG hu 6.333.04 0.48 0.27 IgG mu-anti-huIgG cyno 11.47 6.74 0.59 0.47 PD-1/CTLA-4mu-anti-huIgG hu 2.95 2.36 0.81 0.03 DuetMab mu-anti-huIgG cyno 4.902.15 0.44 0.03 mu-anti-huIgG mu 1.40 693 496 0.12 mu-anti-huIgG hu 6.844.21 0.42 0.08 mu-anti-huIgG cyno 11.43 6.34 0.55 0.18

Reporter Gene Assays

Results from reporter gene assays show that PD-1/CTLA-4 bispecificbinding proteins inhibited the PD-1 and CTLA-4 pathways (FIG. 6A-D). TheBiS5 binding protein retained PD-1 potency compared to parent but had ˜3fold reduced potency compared to anti-CTLA-4 IgGs. The DuetMab antibodyhad a ˜9 fold reduction in PD-1 potency and ˜16 fold reduction forCTLA-4 (compared to IgG4P). There is a need in the art for a moleculethat has reduced CTLA-4 targeting but retains functional activity (e.g.,as shown below in the SEB assay). The PD-1/CTLA-4 bispecific bindingproteins thus have the potential to provide a safety benefit topatients.

Staphylococcal Enterotoxin B (SEB) Assay

Results from an SEB assay show that DuetMab and BiS5Ab had equivalentactivity in SEB primary immune cell assays (FIG. 7), and DuetMab showedgreater activity compared to DummyDuet control arms (FIG. 8A). DuetMabshowed approximately equivalent activity to the combination of parentmolecules, and greater activity compared to LO115 or the CTLA-4 antibodyMEDI1123 (tremelimumab) (FIG. 8B). Finally, BiS5 and DuetMab showedgreater activity compared to a combination of new isotype controls(FIGS. 9A-B). Data were obtained from four donors across two independentexperiments, and these particular assays required the use of IFNγ.

Mixed Leukocyte Reaction (MLR) Assay

MLR assays were performed to test the PD-1/CTLA-4 bispecific molecules.PD-1/CTLA-4 DuetMab and BiS5Ab had equivalent activity in the mixedlymphocyte reaction (MLR) assay (FIGS. 10A-C). PD-1/CTLA-4 DuetMab hadgreater activity compared to the combination of DummyDuet/isotypecontrol arms. (FIGS. 11A-D). PD-1/CTLA-4 DuetMab had about equivalentactivity compared to the combination of parental antibody controls(FIGS. 12A-D). Finally, PD-1/CTLA-4 DuetMab had about equivalentactivity compared to the competitor PD-1/CTLA-4 combination and hadgreater activity than anti-PD-1 alone (e.g., pembrolizumab andnivolumab) (FIGS. 13A-D).

Pharmacokinetic and Pharmacodynamic (PK/PD) Studies

A study was performed to examine single dosepharmacokinetics/pharmacodynamics (PK/PD) in cynomolgus monkeys. Thestudy design is shown in FIG. 14. DuetMab showed clear pharmacodynamics(PD) in cynomolgus monkeys, and robust PD responses were observed forboth molecules (FIGS. 15A-B). Thus confirming the viability of thePD-1/CTLA-4 bispecific binding protein in an in vivo setting,

T Cell Dependent Antibody Response (TDAR)

Cynomolgus monkeys were dosed intravenously (saphenous or cephalic vein)with the indicated dose (0.5, 5, 50 mg/kg) of DuetMab or BiS5 bispecificmolecules. Keyhole limpet hemocyanin (KLH) protein was reconstitutedwith the appropriate amount of sterile water for injection under sterileconditions. Low dose KLH solution was administered subcutaneously oneach animal's back on two occasions (Day 1 and Day 29). Blood samplesfor further analysis were obtained from all animals. Evaluation ofKLH-specific IgM and IgG antibody titers were performed. Anti-KLHantibodies in monkey serum were detected using ELISA.

T cell dependent antibody response (TDAR) was seen in cynomolgus monkeysdosed with PD-1/CTLA-4 DuetMab (FIG. 16A) and PD-1/CTLA-4 BiS5Ab (FIG.16B).

CHO Cells Expressing Diverse Levels of Human PD-1 and/or CTLA-4

A model system was developed to study PD-1/CTLA-4 bispecific moleculesusing stable CHO cells expressing diverse levels of human PD-1 and/orCTLA-4 (FIG. 17). Free antigen binding-arms on cell-bound DuetMab weredetected by flow-cytometry using fluorescently-labeled soluble PD-1 andCTLA-4 proteins. The results of this assay show that PD-1/CTLA-4 DuetMabconcurrently binds PD-1 and CTLA-4 on the surface of the same cell(FIGS. 18A-C).

CTLA-4 is continually endocytosed into clathrin-coated pits, resultingin only a small fraction of the receptor expressed at the cell surfaceat any given time. Recycling of cell-surface CTLA-4 is rapid, with morethan 80% of surface CTLA-4 being internalized within 5 minutes. Thus, anexperiment was performed to address whether co-operative bindingdifferentiates PD-1/CTLA-4 DuetMab over a combination of anti-PD-1 andanti-CTLA-4 antibodies in the saturation of CTLA-4 on cells expressingexcess PD-1 (FIGS. 19A-C). Receptor occupancy of each target antigen wasdetermined independently using labeled anti-PD-1 and anti-CTLA-4 mAbs.

It was found that parental monoclonal antibodies bound and occupiedtheir target receptor without a measurable effect on the untargetedreceptor (FIGS. 20A-D). PD-1/CTLA-4 DuetMab saturated CTLA-4 on CHOcells expressing excess PD-1 at ˜250-fold lower concentrations comparedto a combination of the monoclonal antibodies (FIGS. 21A-D). PD-1/CTLA-4DuetMab saturated CTLA-4 on CHO cells expressing excess PD-1 at˜500-fold lower concentrations compared to cells expressing only CTLA-4(FIGS. 22A-F). The PD-1/CTLA-4 DuetMab preferentially bound in cis toPD-1 and CTLA-4 on the surface of the same cell, as determined byquantitation of doublet formation within total pre-mixed CHO population(FIGS. 23A-B). However, PD-1/CTLA-4 DuetMab can also bind in trans tosingle-expressing cells. PD-1/CTLA-4 DuetMab took on internalizationproperties of the parent anti-CTLA-4 antibody, tremelimumab (FIGS.24A-D). Without being bound by theory, the effect shown by this moleculehas the potential to induce downregulation of PD-1. The internalizationproperties of PD-1/CTLA-4 DuetMab were also seen in stable CHO cellsexpressing 10-fold excess PD-1 (FIG. 25B).

Example 2(b) PD-L1/CTLA-4 Bispecific Binding Proteins

The following bispecific binding proteins that bind PD-L1 and CTLA-4were created using the parental sequences identified above in Table 2.Proteins identified as Bis2, Bis3, and Bis5 were generated with thesequences in Table 6 below and sequences identified below were assessedfor concurrent antigen binding activity using the Octet binding assay asdescribed above in section 2(a) (FIG. 26)

TABLE 6 BIS constructs for PD-L1/CTLA-4 Description Sequence Bis2DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIY PD-L1/CTLA-4AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFG HCCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 14) Bis2EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLI PD-L1/CTLA-4YDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT LCFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) Bis3EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLE PD-L1/CTLA-4WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV HCYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16) Bis3EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLI PD-L1/CTLA-4YDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT LCFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17) Bis5EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLE PD-L1/CTLA-4WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV HCYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 18) Bis5EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLI PD-L1/CTLA-4YDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT LCFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19)

Example 2(c) PD-1/TIM3 Bispecific Binding Proteins

The following bispecific binding proteins (Table 7) that bind PD-1 andTIM3 were created using the parental sequences identified above in Table2. Proteins identified as Bis3, Bis5, and DuetMab were generated withthe sequences identified below and were assessed for concurrent bindingstudies by Octet analysis. Briefly, streptavidin (SA) biosensors(ForteBio) were used to capture biotinylated human TIM3-IgV domain at 2μg/ml in PBS pH 7.2, 3 mg/ml BSA, 0.05% (v/v) Tween 20 (assay buffer).Following a washing step the loaded biosensors were subjected forsuccessive association and dissociation interactions first with samplewells carrying the bispecific antibodies at 200 nM and then with wellscarrying PD-1 antigen at 200 nM. Biotinylated human TIM3-IgV domain wasloaded on Streptavidin sensors followed by sequential interactions firstwith bispecific molecules and then with PD-1 antigen. The bindingresults are shown in FIGS. 27A-27B.

TABLE 7 BIS constructs for PD-1/TIM3 Description SequenceTIM3 WT #62 scFv EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGCGTKLTVL (SEQ ID NO: 20) PD-1 HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE chain + TIM3 (WTWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY #62) scFvYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGCGTKLTVL (SEQ ID NO: 21) BiS5EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1 HeavyWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY chain + TIM3 (WTYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES #62) scFvTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGCGTKLTVLGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K (SEQ ID NO: 22) BiS5QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1 LCPRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) BiS3EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1 HeavyWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY chain + TIM3 (WTYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES #62) scFvTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDHWLFGCGTKLTVL (SEQ ID NO: 24) BiS3QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1 LCPRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) DuetMabEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1 Heavy chainWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) DuetMabQTVLTQPPSVSVAPGKTASISCGGDNIGGKSVHWYQQKPGQAPVLVIY TIM3YDSDRPSGIPQRFSGSNSGNTATLTIHRVEAGDEADYYCQVLDRRSDH LCWLFGGGTKLTVLGQPKAAPSVTLFPPCSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS (SEQ ID NO: 26) DuetMabEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW TIM3 HCVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY KnobCARGSYGTYYGNYFEYWGRGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 27)

Tumor-Specific Killing Activity Assay

Rosenberg Clone-Melanoma Killing Assay.

The general cell killing activity of the TIM3/PD-1 bispecific bindingmolecules and the parental TIM3 antibody were tested using the RosenbergClone: JR6C12 and Melanoma cell line: Mel324.

General Assay Protocol

JR6C12 cells functioned as effectors, and are a human CD8+ T cell lineexpanded from a melanoma patient and specific for gp100-melanomaantigen. To assess therapeutic potential, the Mel624 tumor cells werefluorescently labeled and added with the effectors (JR6C12) andcandidate antibody that binds TIM3 and or PD-1. Cells were coculturedfor 16 hours. The multiple panels in FIG. 28A provide a visualrepresentation that the addition of TIM3 62 either in combination withanti-PD1 or as PD-1/TIM3 bispecific molecules (as described in Table 7)enhance T cell activation and tumor killing.

Furthermore, as shown in FIGS. 28B-28C, PD-1/TIM3 bispecific moleculesdemonstrate the greatest tumor killing potency relative to anti-TIM3,anti-PD-1 or isotype control monotherapy as assessed by (b) tumor cellviability dye uptake and (c) IFN-gamma secretion.

In addition to clone 62, another bispecific binding protein that bindsPD-1 and TIM3 in DuetMab format was created using the parental sequencesidentified above in Table 2. The PD-1/TIM3 DuetMab was generated withthe sequences in Table 8 below. The sequence of the TIM3 arm wasobtained from 013-1, which is an affinity mature variant of clone 62 andthe sequence of the anti-PD-1 arm was obtained from LO115, which isidentical to the PD-1 arm used for the PD-1/CTLA-4 DuetMab bispecificantibody described above. The PD-1(LO115)/TIM3(013-1) bispecificantibody was assessed as discussed below, including in comparison withPD-1/TIM3 BiS3 and BiS5.

TABLE 8 DuetMab construct for PD-1/TIM3 Description Sequence DuetMabQIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA PD-1 (LO115)PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE Amino acidAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7) DuetMabCAGATCGTGCTGACCCAGTCCCCTGCCACCCTGTCCCTGAGCCCTGG PD-1 (LO115)CGAGAGAGCCACCCTGAGCTGCTCCGCCTCCTCCAAGCACACCAAC LCCTGTACTGGTCCCGGCACATGTACTGGTATCAGCAGAAGCCCGGCC Nucleic acidAGGCCCCTCGGCTGCTGATCTACCTGACCTCTAACCGGGCCACCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCCTCCAACCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 8) DuetMabEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE PD-1 (LO115)WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY HCYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG Amino acidGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) DuetMabGAGGTGCAGCTGGTGGAATCCGGCGGAGGACTGGTGCAGCCTGGC PD-1 (LO115)GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACATTCTCCGA HCCTACGGCATGCACTGGGTCCGACAGGCCCCTGGAAAGGGCCTGGAA Nucleic-acidTGGGTGGCCTACATCTCCTCCGGCTCCTACACCATCTACTCCGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACAGCCGTGTACTACTGTGCCAGACGGGCCCCTAACTCCTTCTACGAGTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCTAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCGAGCAGCAAAAGCACCTCTGGTGGCACCGCAGCACTGGGTTGTCTGGTGAAAGATTATTTTCCGGAACCGGTTACCGTTTCTTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTTCTGCAGAGCAGCGGTCTGTATAGCCTGTCTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAATGTGAATCATAAACCGAGCAATACAAAAGTTGATAAACGCGTTGAACCGAAAAGCTGTGACAAAACTCACACGTGCCCACCGTGCCCAGCACCTGAGTTCGAGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTGCACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAGCTGCGCGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 10) DuetMabSYVLTQPPSVSVAPGKTARITCGGDNIGGKSVHWYQQKPGQAPVLVIY TIM3 (O13-1)YDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDRRSDHF LCLFGGGTKLTVLGQPKAAPSVTLFPPCSEELQANKATLVCLISDFYPGAV Amino acidTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS (SEQ ID NO: 28) DuetMabAGCTACGTGCTGACGCAGCCGCCGTCAGTGTCAGTGGCCCCAGGAA TIM3 (O13-1)AGACGGCCAGGATTACCTGTGGGGGAGACAACATTGGAGGTAAAA LCGTGTTCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGTTGGT Nucleic acidCATCTATTATGATAGTGACCGGCCCTCAGGCATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGATTATTACTGTCAGGTGTTGGATCGTCGTAGTGATCATTTCCTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTGCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAAGTGTC A (SEQ ID NO: 29) DuetMabEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW TIM3 (O13-1)VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY HCCARGSYGTYYGNYFEYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGG Amino acidTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSVDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30) BiS3 PD-1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE (LO115)/TIM3WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY (O13-1) HCYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG Amino acidGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGKTARITCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDRRSDHFLFGCGTKLTVL (SEQ ID NO: 89) BiS3 PD-1QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA (LO115)/TIM3PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS (O13-1) LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE Amino acidAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) BiS5 PD-1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE (LO115)/TIM3WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY (O13-1) HCYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG Amino acidGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYGTYYGNYFEYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGKTARITCGGDNIGGKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDRRSDHFLFGCGTKLTVLGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 91)BiS5 PD-1 QIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQA (LO115)/TIM3PRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSS (O13-1) LCNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE Amino acidAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92) DuetMabGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG TIM3 (O13-1)GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGC HC-TATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT Nucleic acidGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGGTCCTATGGTACCTACTACGGAAACTACTTTGAATACTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGTGCGTCGACCAAAGGTCCGAGCGTGTGCCCGCTGGCACCGAGCAGCAAAAGCACCTCTGGTGGCACCGCAGCACTGGGTTGTCTGGTGAAAGATTATTTTCCGGAACCGGTTACCGTTTCTTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTCCTGCAGAGCAGCGGTCTGTATAGCCTGTCTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAATGTGAATCATAAACCGAGCAATACCAAAGTTGATAAACGCGTTGAACCGAAAAGCGTGGACAAAACTCACACGTGCCCACCGTGCCCAGCACCTGAGTTCGAGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATGCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCTTAAGCCTGTCTCCGGGTAAA (SEQ ID NO: 31)

Octet Binding Assay (DuetMab, TIM3 Arm Affinity Mature Variant)

Concurrent binding studies to two distinctive antigens, PD-1 and TIM3,were performed by Octet analysis. Biotinylated human TIM3 was loaded onStreptavidin sensors followed by sequential interactions first withPD-1/TIM3 DuetMab and then with soluble PD-1 antigen. Streptavidin (SA)biosensors (ForteBio) were used to capture biotinylated human TIM3 at 5μg/ml in PBS pH 7.2, 3 mg/ml BSA, 0.05% (v/v) Tween 20 (assay buffer).Following a washing step the loaded biosensors were subjected forsuccessive association and dissociation interactions first with samplewells carrying DuetMab PD-1/CTLA-4 bispecific antibody having the TIM3arm (O13-1), which is the affinity mature variant of clone 62 TIM3antibody, was loaded at 200 nM and then with wells carrying human PD-1antigen at 200 nM. The binding results are shown in FIG. 29.

Intrinsic kinetics of the PD-1/TIM3 DuetMab bispecific antibody was alsoassessed via BiaCore. Binding experiments were carried out using aBIAcore T200 instrument (BIAcore). To capture the antibody, mouseanti-hulgG-Fab was immobilized on a CM5 chip to a target response of2000 RU. 100 nM of the DuetMab or mAbs were flowed at 20 μL/min for 5min to achieve approximately 100 response units of captured antibody.Antigen were then injected serially at a flow rate of 50 μl/min for 5min. Kinetic parameters (k_(on) and k_(off)) and dissociation constant(KD) were calculated from a non-linear fit using BIAevaluation 4.1software. The binding results are shown in Table 9.

TABLE 9 BiaCore data for PD-1/TIM3 capture TIM3 ka (M⁻¹s⁻¹) kd (s⁻¹)K_(D) (nM) capture PD-1 ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) O13-1 Human1.88E+06 7.52E−03 4.01 LO115 Human 3.02E+05 2.37E−04 0.79 IgG1-TMIgG1-TM DuetMab Human 1.96E+06 8.13E−03 4.15 DuetMab Human 2.95E+052.36E−04 0.69 O13-1 Cyno 2.79E+06 7.18E−02 25.69 LO115 Cyno 3.46E+052.38E−04 0.81 IgG1-TM IgG1-TM DuetMab Cyno 2.98E+06 7.61E−02 25.57DuetMab Cyno 4.90E+05 2.15E−04 0.44

PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, and DuetMab,bound to CHO cells overexpressing human TIM3 or human PD-1 (FIG. 30 andTable 25), PD-1 and TIM3 expression (DMF4) are shown in FIG. 31.

TABLE 25 anti-TIM3 LOO131 anti-PD1 LO115 Duetmab Bis3 Bis5 Nip228 HuIgG1TM EC50 0.003968 NA 0.1036 0.02853 0.01603 NA R square 0.9951 0.97920.9982 0.9964 0.9954 0.9804

CMV Ag Recall Assay

PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, and DuetMab,enhanced CD8+ T cell proliferation in a CMV antigen recall assaycompared to isotype treatment (FIGS. 32A-C).

Mixed Leukocyte Reaction (MLR) Assay

PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, and DuetMab,enhanced interferon (IFNγ) secretion in the mixed lymphocyte reaction(MLR) assay, with activity trending above mono- and combination therapy(FIGS. 33A-D).

PD-1/TIM3 bispecific antibodies, including BiS3, BiS5, and DuetMab,demonstrated similar activity as parental LO115 IgG1 in a jurkat NFκBreporter line that predominantly expresses PD-1 (87% PD-1 singlepositive) (FIGS. 34A-C).

In summary, three bispecific formats (DuetMab, Bis3, and Bis5) weregenerated for PD-1/TIM-3. All bispecific formats show in vitrofunctionality equivalent to or better than anti-PD-1, suggesting thesemolecules may provide superior advantage to current immuno-oncologystrategies.

Example 2(d) OX40/PD-L1 Bispecific Binding Proteins

The following bispecific binding proteins that bind PD-L1 and OX40 werecreated using the parental sequences identified above in Table 2.Proteins identified as Bis2, Bis3, and Bis5 were generated with thesequences in Table 10 below and were assessed for concurrent antigenbinding activity using the Octet binding assay as discussed below.

TABLE 10 BIS constructs for OX40/PD-L1 Description SequenceOX40 LCv kappa DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32) OX40SLR LCv kappaDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93) BiS2-EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLI PD-L1-OX40 HC-YDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT 4PFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 34) BiS3-QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYI OX40 HC-4P-PD-L1GYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS (SEQ ID NO: 35) BiS5-QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYI OX40 HC-4P-PD-L1GYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 36)BiS5-OX40SLR HC- QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIG1-N434A-PD-L1 GYISYNAITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYEGGHAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGCGTKVEIKGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSP GK (SEQ ID NO: 94)

Octet Binding Assay

To evaluate binding of the bispecific binding molecules disclosedherein, an Octet QK equipped with Ni-NTA biosensor tips and 10× kineticsbuffer were used (ForteBio, Menlo Park, Calif.). For this particularseries of bispecific binding proteins, His-tagged PD-L1-Fc, his-taggedPD-1-Fc and hOX40-Fc (human recombinant proteins) were purchased fromR&D Systems (Minneapolis, Minn.). All binding assays were performed at25° C.

Sample plates were agitated at 1000 rpm prior to analysis. The Ni-NTAbiosensor tips were pre-wetted for 5 min. in 1× kinetic buffer. The 1×kinetic buffer also served as the running buffer for baselinedetermination and as the dilution buffer for antigens and bispecificantibodies. Ni-NTA biosensor tips were dipped into 100 nM his-taggedPD-L1-Fc (see, (b), below) or his-tagged PD-1-Fc for antigen capture forabout 1 min. The antigen-coated biosensor tips were each dipped into 10μh/ml bispecific antibodies for ˜5 minutes and then moved into a columnof wells containing 100 nM hOX40-Fc antigen for 2 minutes. The bindingresults show that BiS2/BiS3 OX40Ab/PD-L1 molecules bind to bothPD-L1-His and hOX40-Fc, and that BiS2 OX40Ab/PD-L1 binds with greateraffinity than BiS3 OX40Ab/PD-L1. BiS2 PD-1/OX40 was used as a control(FIG. 35).

Staphylococcal Enterotoxin B (SEB) Assay

An SEB assay using the protocol described above showed that theOX40/PD-L1 bispecific molecule was active in both BiS2 and BiS3 formats(FIGS. 36A-B).

PD-L1 Reporter Assay

Materials:

-   -   Cell lines and culture conditions:    -   Human PD-1 Jurkat NFAT luciferase clone 2 reporter    -   PD-L1 expressing CHO scFv OKT3 (UBC) (All cells were maintained        in RPMI 1640 media plus 10% FBS and 1× pen/strep antibiotics        (RPMI complete media) at 37° C. in a humidified tissue culture        incubator).    -   RPMI-1640, LifeTechnologies cat# A1049101    -   Heat inactivated newborn calf serum (FBS), LifeTechnologies        cat#26010074    -   Complete RPMI medium: RPMI-1640 plus 10% FBS    -   100× Penicillin/Streptomycin, LifeTechnologies cat#15140-122    -   96 well TC treated flat bottom culture plates, Costar 3903, VWR        cat#29444-010    -   SteadyGlo Luciferase Assay System, Promega, cat#E2510    -   Test Antibodies    -   EnVision Multilabel Plate Reader, Perkin Elmer

Methods:

For 2-cell bioactivity assay for neutralization of PD-L1 inhibition,PD-L1 expressing CHO scFv OKT3 cells were trypsinized, neutralized withwarm RPMI complete media, and collected in a 50 mL conical tube. Cellswere pelleted at 380 g for 5 min at RT, and then suspended in fresh RPMIcomplete media and counted on a Vi cell counter. PD-L1 expressing CHOscFv OKT3 cells were adjusted to 0.4e6/mL and 25 μL (10,000 cells) perwell were plated as shown on the plate layout. Cells were allowed toadhere to plates for 3 hours. Thereafter, 50 μL of RPMI containing testreagents (2× final conc) were aliquoted onto the CHO cells and incubatedfor an additional 1 hour. This incubation gives the test reagent time tobind to PD-L1 on the surface of CHO cells. At 1 hour, PD-1 expressingJurkat NFAT luciferase reporter cells were collected in a 50 mL conicaltube, pelleted at 380 g for 5 min at RT, and re-suspended in fresh, warmRPMI complete media. Cells were adjusted to 1.2e6/mL and 25 μL (30,000)cells plated into wells with PD-L1 expressing CHO scFv OKT3 cells andtest articles.

Cells and test reagent were further incubated for 18 hours for PD-1Jurkat reporter cell activation. Thereafter, SteadyGlo luciferasereagent was prepared and 100 μL aliquoted to each well. Complete lysiswas achieved by gentle shaking at RT (200 rpm orbital shaker) for 15min. After lysis, luciferase activity was measured on an EnvisionMultilabel Plate Reader using the US96 luminescence protocol. LuciferaseRLU was plotted versus log [test reagent] in Graphpad Prism software,and EC₅₀ values for PD-L1 antagonism determined using non-linearregression analysis, 4-parameter fit of sigmoidal dose-response curves.

Results:

OX40/PD-L1 BiS2/3 were tested against PD-L1/PD-1 parents and NIP228(G4P)controls using a five-point dose titration with a starting point of 100nM (PD-L1). OX40-PD-L1 BisAbs were both shown to be active and havestronger agonism than PD-L1(4736) parent (FIG. 37) The BiS2 and BiS3formats performed similarly.

CMV Ag Recall Assay

In the CMV Ag recall assay (using the protocol described above), theBiS2 and BiS3 molecules demonstrated equal activity relative tocombination (FIG. 38).

All the binding and immune response assays discussed above provideillustrative data that the bispecific binding molecules disclosed hereinexhibit specific binding for both target molecules—in some instancesgreater binding activity than the combination of individual monospecificparental binding molecules (antibodies), and can induce or enhance animmune response. Furthermore, the molecules are shown to have cellkilling activity against a cancer cell line. As such, the data showsthat these molecules and bispecific platform structure(s) representexcellent candidates for immuno-oncology therapeutics.

Octet Binding Assay (OX40(SLR)/PD-L1 BiS5)

To evaluate binding of the bispecific binding molecules disclosedherein, an Octet QK equipped with Ni-NTA biosensor tips and 10× kineticsbuffer were used (ForteBio, Menlo Park, Calif.). For this particularseries of bispecific binding proteins, His-tagged PD-L1-Fc, his-taggedPD-1-Fc and hOX40-Fc (human recombinant proteins) were purchased fromR&D Systems (Minneapolis, Minn.). All binding assays were performed at25° C. The binding results show that BiS5 OX40Ab/PD-L1 molecules bind toboth PD-L1-His and hOX40-Fc (FIG. 39).

PD-L1/OX40 BiS5 bound to CHO cells expressing CHO cells expressing humanor cynomolgus OX40 and PD-L1/B7H1 (FIGS. 40A-F). Binding of PD-L1/OX40BiS5 constructs was also measured by flow cytometry (HyperCyt) (FIG.42). OX40 IgG4P and OX40/PD-L1 bispecifics bound to Jurkat OX40 reportercells. PD-L1 IgG and OX40/PD-L1 bispecifics bound to NCI H358 and CHOK1B7H1(PD-L1)/OKT3 cells. All IgG and bispecifics bound to HEK CD32acells.

PD-L1 and OX40 Reporter Assay

In the PD-L1 reporter assay (using the protocol described above), allPD-L1 scFv containing bispecifics and positive control IgGs displayedactivity (FIGS. 42A-B). The single arm OX40 controls and isotypecontrols did not display any activity in this assay. The EC₅₀ values andhill slopes are consistent with values obtained in previous assays forthe anti PD-L1 parental controls and the PD-L1 Bis2, Bis3 and Bis5constructs.

In an OX40 reporter gene assay using HEK CD32a cells, the bispecificconstructs had equal activity to each other, and Fc mediated agonism wasobserved (FIGS. 43A-B). OX40/PD-L1 Bis5 N434A IgG1 had equivalent EC₅₀activity to OX40 IgG4P and MEDI0562 (OX40 IgG1).

In an OX40 reporter gene assay using CHOK1 PD-L1 over expressing cells,the OX40/PD-L1 bispecifics show equal agonism (FIGS. 44A-B). OX40/PD-L1Bis5 N434A IgG1 had equivalent EC₅₀ activity to other Fc variants of theOX40/PD-L1 Bis5 bispecific Mab tested. No agonism with OX40 IgGs orPD-L1 IgGs was detected. Thus, PD-L1 mediated OX40 agonism wasdemonstrated.

PD-L1 mediated OX40 agonism with tumor cells using OX40/PD-L1 bispecificmolecules was detected (FIGS. 45A-B). OX40/PD-L1 bispecific moleculesshowed equal agonism in this assay—bell shaped curves. No agonism withOX40 IgGs was observed, therefore demonstrating a benefit of usingbispecifics over OX40 IgG plus PD-L1 IgG combination. No agonism wasseen with NCI H358 PD-L1 KO cells (FIGS. 46A-D) showing that NCI H358agonism seen with cells is PD-L1 specific.

Staphylococcal Enterotoxin B (SEB) Assay

In the SEB assay, OX40/PD-L1 bispecific molecules had greater activitythan the combination of individual antibodies to OX40 and PD-L1 (FIGS.47A-D). In particular, the G4P construct had greater activity than theG1 construct. Wild-type, a YTE containing variant, and the N434A varianthad equivalent activity.

Treg Suppression Assay

A Treg suppression assay was performed to test the OX40/PD-L1 bispecificmolecules (FIGS. 4A-D). OX40/PD-L1 bispecific molecules were active onCD4+ T_(eff) only in the presence of PD-L1 (FIGS. 49 and 50). Withoutbeing bound by theory, this indicated crosslinking of OX40 in trans.OX40/PD-L1 bispecific molecules suppressed T_(reg) inhibitory effects,but only when crosslinked by binding to plate-immobilized PD-L1.

Mixed Leukocyte Reaction (MLR) Assay

A MLR assay was performed to test the OX40/PD-L1 bispecific molecules(FIGS. 51A-B). OX40/PD-L1 bispecific molecules had greater activity thanthe combination of individual antibodies to OX40 and PD-L1 (FIG. 52A-E).

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay

An ADCC assay was performed to test the OX40/PD-L1 bispecific molecules.ADCC assay using freshly isolated NK cells as effector cells and CHOK1PD-L1 B7H1 and CHOK1 OX40 overexpressing cells, respectively, as targetcells at an effector to target (E:T) ratio of 20:1. Target cell lysiswas analyzed using release of Europium from labelled target cells after5 hours. In the ADCC assay, OX40/PD-L1 BiS2 and BiS5 mediated ADCCagainst PD-L1 or OX40 expressing CHO cells (FIGS. 53A-B and 54).

A CD107a mobilisation assay was performed using freshly isolated NKcells as effector cells and PD-L1 and OX40 overexpressing CHO K1 astarget cells at a E:T ratio of 10:1. CD107a mobilisation to the cellsurface of NK cells was analysed by flow cytometry after 4 hours. BiS2and BiS5 OX40/PD-L1 bispecific molecules increased CD107a mobilizationof NK cells against PD-L1 and OX40 expressing CHO cells inantibody-dependent cell-mediated cytotoxicity (ADCC) assays (FIG. 55).BiS2 and BiS5 OX40/PD-L1 bispecific molecules increased CD107amobilization of NK cells against activated allogeneic T cells that haveup-regulated OX40 and PD-L1 (FIG. 56). BiS5 OX40/PD-L1 increased CD107amobilization of NK cells from two different donors against activatedallogeneic T cells (FIGS. 57A-B).

Pharmacokinetic and Pharmacodynamic (PK/PD) Studies

A study was designed to compare the PK/PD of OX40/PD-L1 bispecificmolecules (FIG. 58). Serum concentration time profiles for PD-L1/OX40bispecific molecules were compared in cynomolgus monkeys (FIG. 59 andTable 11). Mean T1/2 for the Bis5 OX40/PD-L1 IgG1 N434A molecule wasgreater than for the WT Bis5 molecule; clearance rate was less for theBis5 OX40/PD-L1 IgG1 N434A molecule as compared to the WT Bis5 molecule.Both molecules similarly reduced soluble PD-L1 in the serum, andelicited a significant increase in the percentage of Ki67+ total memoryCD4+ T cells, total memory CD8+ T cells and NK cells.

TABLE 11 Pharmacokinetic parameters of BiS5-OX40/PD-L1-G1 AUClast AUCinf% AUCExtrap C0 Cmax Test Article Animal (ng*h/mL) (ng*h/mL) (%) (ng/mL)(ng/mL) BiS5- 2028 5490000 6840000 19.8 82900 82300 OX40/PD- 20336530000 10800000 39.7 77700 79100 L1-G1 2059 6510000 9260000 29.7 9450089800 N434A Mean 6180000 8980000 29.7 85000 83700 SD 597000 2000000 9.918610 5520 BiS5- 2032 4260000 4530000 6.08 104000 101000 OX40/PD- 20473670000 3900000 5.84 131000 105000 L1-G1 WT 2056 4190000 4340000 3.4574400 74400 Mean 4040000 4260000 5.13 103000 93300 SD 320000 324000 1.4528200 16500 Tmax Cl Vss T½ Test Article Animal (h) (mL/min/kg) (L/kg)(h) Rsq BiS5- 2028 2 0.0122 0.0773 70 0.836 OX40/PD- 2033 6 0.00770.0842 128 0.873 L1-G1 2059 2 0.00899 0.0746 97.1 0.933 N434A Mean 3.330.00963 0.0787 98.3 0.881 SD 2.31 0.0023 0.00496 28.9 0.049 BiS5- 2032 20.0184 0.0596 37.9 0.952 OX40/PD- 2047 2 0.0214 0.0672 44.6 0.987 L1-G1WT 2056 2 0.0192 0.0555 34 0.99 Mean 2 0.0196 0.0608 38.9 0.976 SD 00.00154 0.00593 5.35 0.0211

OX40/PD-L1 bispecific molecules reduced serum soluble PD-L1concentrations below the assay LLOQ (FIG. 60). The N434A mutationimproved pharmacokinetics of BiS5-OX40/PD-L1-G1. In particular, CL wasreduced by approximately half; there was a corresponding 2-fold increasein T1/2 and AUCinf; and Cmax and Vss not impacted. This was consistentwith previously reported effects of this mutation on the PK ofmonoclonal antibodies. Thus, progress was made towards mAb-like PK forBiS5-OX40/PD-L1-G1 IO BisAb. Serum concentrations of BiS5-OX40/PD-L1-G1BiSAbs were below the limit of quantitation (BLOQ) at 2 weeks, and mayhave been related to ADA.

Substantial and statistically significant increases were seen in totalmemory CD4, total memory CD8, and NK cell proliferation (percentage ofKi67+ cells) comparing PD-L1 OX40 Bis5 groups to control (anti-PcrV-Pslcontrol) Ab group (FIGS. 61A-F). A trend towards significant differenceswas seen between PD-1 LO115 and PD-L1 OX40 Bis5 groups in total memoryCD4, total memory CD8, and NK cell proliferation (Ki67+). There were nostatistically significant differences in proliferation between PD-L1OX40 Bis5 N434A (half-life extended) version and the G1 version. PD-L1OX40 Bis5 N434A and IgG1 versions are biologically active bispecificmolecules.

Example 2(e). OX40/PD-1 Bispecific Binding Proteins

The following bispecific binding proteins that bind PD-1 and OX40 werecreated using the parental sequences identified above in Table 2.Proteins identified as Bis2 and Bis3 were generated with the sequencesin Table 24 below and were assessed for concurrent antigen bindingactivity using the Octet binding assay as discussed below.

TABLE 24 BIS constructs for OX40/PD-1 Description Sequence PD1 LCv kappaQIVLTQSPATLSLSPGERATLSCSASSKHTNLYWSRHMYWYQQKPGQAPRLLIYLTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 97) BiS2-DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIY PD-1-OX40 HC-4PYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKCLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 95) BiS3-EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLE OX40 HC-4P-PD-1WVAYISSGSYTIYSADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRAPNSFYEYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKCLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS (SEQ ID NO: 96)

PD-1/OX40 BiS2 monoclonal antibody (mAb) is a bispecific antibody (FIG.62; PD-1 binding proteins depicted in gray and OX40 binding proteinsdepicted in light gray) engineered to bind concurrently to human andcynomolgus monkey PD-1 and human and cynomolgus monkey OX40. Withoutbeing bound by theory, a proposed mechanism of action suggests dualsignaling effects on T cells after binding in cis to both OX40 and PD-1,agonism of the T cell co-stimulatory surface receptor OX40, and blockadeof immunosuppressive PD-1 (FIG. 63).

Octet Binding Assay

Concurrent binding activity was shown for two different lots of thePD-1(LO115)/OX40 BiS2 mAb to PD1-His and human OX40-Fc (FIG. 64).

OX40 Reporter Assay

PD-1(LO115)/OX40 BiS2 mAb showed activity comparable to other OX40agonists (FIGS. 65A-B). Proteins were stored at 4° C. and usedimmediately, freeze/thawed three times, stored at 4° C. for 7 days andstored at 40° C. for 7 days. Activity was reported as relative lightunits versus concentration of mAb. EC₅₀ was ˜2 nM for PD1(LO115)/OX40BiS2 mAb at 4° C. on day 0.

PD-1/PD-L1 Reporter Assay

PD-1(LO115)/OX40 BiS2 mAb showed activity comparable to other PD-1agonists (FIGS. 66A-B). Proteins were stored at 4° C. and usedimmediately, freeze/thawed three times, stored at 4° C. for 7 days andstored at 40° C. for 7 days. Activity was reported as relative lightunits versus concentration of mAb. EC₅₀ was ˜1 nM for PD1 (LO115)/OX40BiS2 mAb at 4° C. on day 0. Two sets of primary human in vitro potencyassays have been conducted; an antigen-recall T cell assay and a T cellco-stimulation using staphyloccal entertoxin B (SEB).

Staphylococcal Enterotoxin B (SEB) Assay

In the SEB assay, PD-1(LO115)/OX40 BiS2 mAb induced an increase in thelevels of IL-2 detected in the supernatant of cells after 3 days inculture (FIG. 67). Thus, PD-1/OX40 BiS2 mAb can concurrently bind to itshuman target antigens and can co-stimulate T cells in vitro.

In the antigen-recall assay PD-1/OX40 BiS2 mAb drove an increase in thelevels of interferon (IFN)-gamma as compared to the parent mAbs and thecombination of the parent mAbs (FIGS. 68 and 69).

CMV Ag Recall Assay

Results of the CMV Ag recall assay (using the protocol described above),the BiS2 and BiS3 molecules did not demonstrate equal activity relativeto combination (FIG. 70). The data show that PD-1/OX40 BiS2 IgG4P mAb isactive in vitro and in vivo. PD-1/OX40 BiS3, which differs in structurefrom PD1/OX40 BiS2, was not detectably active. Thus, PD-1/OX40 BiS3 (notactive) is different from BiS2 (active).

Pharmacokinetic and Pharmacodynamic (PK/PD) Studies

Cynomolgus monkey was considered to be a pharmacologically relevantnonclinical species to test the functional activity of PD-1/OX40 BiS2mAb. The pharmacokinetics (PK) and pharmacodynamics (PD) of PD-1/OX40BiS2 mAb were assessed in a non-GLP (Good Laboratory Practices) study incynomolgus monkeys. PD-1(LO115)/OX40 BiS2 mAb PK and PD (percent Ki67positive CD4+ and CD8+ total memory T cells) were evaluated incynomolgus monkeys (n=3; males) following a single intravenous (IV) doseover the dose range of 0.1 mg/kg to 30 mg/kg. PBMC were collectedpre-dose and on day 1, 8, 11 and 15 post-dose, cryopreserved and thawedbefore being analyzed by flow cytometry. In summary, PD1(LO115)/OX40BiS2 mAb displayed approximately linear PK with a short half-life of0.6-1.7 days (FIG. 70; Table 12).

TABLE 12 Mean pharmacokinetic parameters of PD1(LO115)/OX40 BiS2 mAb.Dose C_(max) AUC_(last) AUC_(INF) CL T_(1/2) Vss (mg/kg) (ug/mL)(day*ug/mL) (day*ug/mL) (mL/day/kg) (day) (mL/kg) 0.1  2.0 (0.5)  1.6(0.3)  1.7 (0.3) 60.2 (9.0) 0.6 (0.1) 47.4 (18.3) 1 25.1 (3.1) 24.8(7.5) 25.1 (7.3)  41.8 (10.4) 0.9 (0.3) 43.2 (17.4) 10 211 (19)  203(9.2)  207 (18.0) 48.6 (4.2) 1.7 (0.2) 72.7 (8.8)  30 607 (83) 576 (49)577 (61) 52.3 (5.5) 1.5 (0.2) 85.6 (4.3)  Values are presented as Mean(Standard Deviation). AUC_(last) = area under the concentration timecurve up to the last measurable concentration; AUC_(INF) = area underthe concentration time curve up to infinite time; C_(max) = maximumobserved concentration; CL: systemic clearance; T_(1/2) = half-life;Vss: terminal phase volume of distribution; V_(ss): Steady-state volumeof distribution.

Mean peak concentrations (C_(max)) increased approximately doseproportionately from 2.0 μg/mL at 0.1 mg/kg to 607 μg/mL at 30 mg/kg.AUC∞ increased approximately dose-proportionally from 1.7 μg·day/mL at0.1 mg/kg to 577 μg·day/mL at 30 mg/kg. Mean serum clearance ranged from41.8 mL/day/kg to 60.2 mL/day/kg. The steady-state volume ofdistribution ranged from 43.2 mL/kg to 85.6 mL/kg. PD results (FIG. 71)showed a dose-dependent increase in CD4+ total memory T cellproliferation (Ki67) and an increase in CD8+ total memory T cellproliferation (Ki67). A representative standard curve for quantitationof PD-1/OX40 in cynomolgus monkey serum is shown (FIG. 72).

Example 3. Physical and Chemical Stability of BiSAb Constructs

A series of experiments were performed in order to evaluate and assessthe physical and chemical stability of the BiSAb constructs as describedherein, relative to other bispecific binding protein structuralstrategies and platforms. In particular, the series of stability studiesdiscussed below identified and analyzed the effect of various pH rangeson stability of the BiSAbs (e.g., hydrolysis, fragmentation,aggregation, thermal stability). As the data show, for the differentillustrative embodiments of the various BiSAb formats, the BiSAbdisclosed herein (identified as “BiS5” in the studies below, and in D/Hformat as shown in Table 13) demonstrated unexpected and surprisingphysical and chemical stability relative to all the other BiSAbstructural motifs.

TABLE 13 BiS5 Stability study constructs Construct Sequence DEVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMHWVRQATGKGLE IgG: LC10WVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVY scFv: 2F4YCARDRYSPTGHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKST SN-(scFv)-GSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS Amino acidSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSYLGWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYQNWPLLTFGCGTKLEIK GGGGSGGGGSGGGGSGGG GSEVQLLESGGGLVQPGGSLRLSCAASGFTFSPYMMQWVRQAPGKCLEWVSSIWPSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRRGGATDYWGQGTLVTVSS GGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 37)V regions are underlined, linkers are in italics. Sequence is as follows:LC10VH, CH1, hinge, CH2, CH3 (N-term), L1-2F4VL-linker-2F4VH-L2, CH3 (C-term) H EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYMMQWVRQAPGKGLEIgG: 2F4 WVSSIWPSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV scFv: LC10YYCARVRRGGATDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT SN-(scFv)-GAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT Amino acidVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCKQYADYWTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMHWVRQATGKCLEWVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTVTVSS GGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 38)V regions are underlined, linkers are in italics. Sequence is as follows:2F4VH, CH1, hinge, CH2, CH3 (N-term), L1-LC10VL-linker-LC10VH-L2, CH3 (C-term)

Example 3.1

Further comparison was performed between the BiS format disclosed herein(“BiS5”) and another BiS format that includes two binding domains (scFvdomains) linked at the hinge region (i.e., between the Fc and Fabregions), identified as “BiS4”. The BiS4 and BiS5 proteins wereexpressed in Chinese hamster ovary (CHO), and purified by routinechromatographic methods. As noted above, these two formats have similarFab and scFv sequences, with the primary difference between them beingthe location of the scFv domain (for BiS4, the scFv is located in thehinge region; for this particular BiS5, a scFv is located in the SNGloop in the C_(H)3 domain, as discussed herein). The purified BiSmolecules were supplied in PBS buffer and protein concentration wasdetermined using NanoDrop ND-1000 (Thermo Scientific, Wilmington, Del.)using an extinction coefficient of 1.54 M⁻¹ cm⁻¹.

pH Screen and Short-Term Stability Study

For pH screen studies, BiS4 and BiS5 antibodies were concentrated to ˜12mg/mL and dialyzed against 6 different pH conditions, 20 mM sodiumsuccinate (pH 5.0), histidine/histidine HCl (pH 5.5, 6.0, and 6.5), andsodium phosphate (pH 7.0 and 7.5), all containing 240 mM sucrose.Dialysis was performed by using Slide-A-Lyzer dialysis cassettes (10 kDamolecular weight cutoff (MWCO), Thermo-Fisher, Rockford, Ill.). Aftercompletion of the dialysis, 0.02% polysorbate 80 was spiked and thefinal concentration of the protein was adjusted ˜10 mg/mL. The BiS4 andBiS5 formulations were sterilized using a 0.22 μm filter (Millipore,Billerica, Mass.) in a pre-sanitized laminar flow hood. One milliliteraliquots were dispensed into 3 mL borosilicate glass type I vials (WestPharmaceutical Services, Exton, Pa.). Samples were stored at 40° C. andanalyzed by SEC at time zero and after storage for 1, 2, 3, and 4 weeks.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry thermograms for time zero samples wereobtained using a VP-Capillary DSC connected to a temperature regulatedauto-sampler (Malvern Instruments Ltd., Westborough, Mass.). To acquirethe thermograms protein concentration of 1 mg/mL was used along with ascan rate of 90° C./h over the temperature range 20° C.-100° C. Thethermograms of BiS4 and BiS5 in different pH conditions ranging from 5.0to 7.5 were buffer subtracted and baseline corrected. Data analysis wasperformed using the DSC plug-in for the Origin 7 SR4 software package.The experimental results were fit to a multistate model with threetransitions to calculate the melting temperature (T_(m)) values. Thepoint where the heat capacity (C_(p)) value for the first thermaltransition reached 500 cal mol⁻¹° C.⁻¹ was considered as onsettemperature (T_(onset)).

High-Performance Size-Exclusion Chromatography (HP-SEC)

To separate aggregate and fragment species from monomer based on size,stability samples were analyzed using an Agilent high-performance liquidchromatography system with a photodiode array detector capable ofrecording 200-400 nm UV absorbance spectra with a 7.8×30 cm², 5 μm,250A, Tosho TSKgel G3000SWxl (TOSOH Biosciences, King of Prussia, Pa.)and a corresponding guard column. To separate the species a mobile phasecontaining 0.1M sodium phosphate dibasic anhydrous, 0.1M sodium sulfate,0.01% sodium azide, pH 6.8, and a flow rate of 1 mL min⁻¹ were used. Theamount of protein injected was about 250 μg. The separation of BiS4 andBiS5 was monitored using the absorbance spectrum of 280 nm. The peakareas for soluble aggregates (multimer and dimer), monomer, andfragments were quantified. Then, the percentage of each of the specieswas calculated and plotted against incubation time to develop kineticplots. pH profile curves for the rate of monomer loss, fragmentation andaggregation per month were developed by calculating the slop of eachkinetic plot.

Thermal Stability of BiS4 and BiS5

The effect of pH on thermal stability of BiS4 and BiS5 was evaluatedthrough analysis of the thermograms obtained by using capillary DSC, andgenerated at six different pH conditions. FIGS. 74A and 74B showsoverlays of DSC thermograms of BiS4 and BiS5, respectively, from pH 5.0to 7.5. As shown in FIG. 73, each thermogram shows three thermalunfolding events with transition temperatures T_(m)1, T_(m)2, andT_(m)3. The first transition (T_(m)1) likely to be associated with thesimultaneous unfolding of the C_(H)2 and scFv domains whereas the second(T_(m)2) and third (T_(m)3) transitions are associated with theunfolding of the C_(H)3 and Fab domains. For both the formats, anincrease in T_(onset), T_(m)1, T_(m)2 and T_(m)3 were observed withincrease in pH up to 6.5 (FIGS. 73A, 73B, 73E and Table 14, below). ForBiS4 and BiS5, no differences were observed in T_(onset), T_(m)2, andT_(m)3 at all pH conditions (FIG. 73E and Table 15) indicating that thepresence of scFv either in the hinge region or C_(H)3 domain does notimpact thermal stability of C_(H)3 and F_(ab). Interestingly, a slightincrease in T_(m)1 was observed for BiS5 at all pH conditions indicatingan increase in thermal stability of either scFv, C_(H)2, or both whenscFv is located in the C_(H)3 domain.

TABLE 14 Effect of pH on thermal onset temperature (T_(onset)) andthermal melting temperatures (T_(m)1, T_(m)2, and T_(m)3) for BiS4 andBiS5 as measured by capillary DSC. BiS4 BiS5 pH T_(onset) T_(m)1 T_(m)2T_(m)3 T_(onset) T_(m)1 T_(m)2 T_(m)3 5.0 56.3 67.8 81.9 84.1 55.9 69.581.8 83.9 5.5 56.8 67.8 81.6 83.8 57.0 69.4 81.7 83.7 6.0 59.3 69.3 83.185.3 59.4 70.7 83.1 85.2 6.5 60.6 70.3 83.8 86.0 60.6 71.6 83.8 85.9 7.060.6 70.2 82.2 84.7 59.9 71.2 81.5 84.3 7.5 60.3 70.0 81.5 84.2 59.671.0 81.0 83.9

Physical and Chemical Stability of BiS4 and BiS5

The physical and chemical stability of the BiS4 and BiS5 formats atdifferent pH values (ranging from 5.0 to 7.5) was evaluated at 40° C.for up 4 weeks. HP-SEC chromatograms at “time zero” were used to comparethe total area, monomer, aggregate, and fragment content of HP-SECchromatograms for other time points. Representative chromatograms ofBiS4 and BiS5 at pH 7.5 time zero compared to 4 weeks are shown in FIG.74A. All the samples contain primarily monomer with low levels ofsoluble aggregates and with or without fragments. At time zero (solidlines), majority of the sample is monomer, with no other notabledifferences except a small difference in the peak height between the twosamples likely due to a slight difference in the concentration (FIG.74A). Dotted lines show an overlay of HP-SEC chromatograms of both theformats in the same pH conditions after storage for 4 weeks at 40° C.Under accelerated temperature stress condition both the formats show anadditional peak, early-eluting peak (multimeric species), a decrease inthe monomer, and elevated levels of fragments (FIG. 74A). The loss ofmonomer due to fragmentation was more prominent in BiS4 compared to BiS5indicating that the BiS5 is more chemically stable. Based on theirstructure, possible fragmentation sites, and retention time, wespeculate that the small fragment peak (RT ˜10.8 min) is a Fab, andlarge fragment peak (RT ˜9.8 min) and the shoulder peak (RT ˜8.7 min) isa Fab with scFv and its corresponding higher molecular weight fragment(HMWF) with Fab, scFv, and Fc, respectively.

To better evaluate the effect of the location of scFv on the physicaland chemical stability of BiS4 and BiS5, percent total area for eachspecies were plotted in a bar chart for time zero and 4 weeks at 40° C.for pH 7.5 (FIG. 74B). As shown in FIG. 74B, at time zero, the monomerpurity for BiS4 and BiS5 are similar. Samples incubated at 40° C. for upto 4 weeks showed significant differences in the type and extent offragments formed. For BiS4 samples, 11.8%, 7.2%, and 3.5% of shoulderpeak (RT ˜8.7 min), large fragment (RT ˜9.8 min) and small fragment (RT˜10.8 min) were formed, respectively (FIG. 74B). Surprisingly, BiS5sample showed only 1.4% of small fragment (RT ˜10.8 min) likely due totethering of scFv from both the sides of the domain to Fc (FIG. 89).

FIGS. 75A-75C show the kinetics of aggregation, fragmentation andmonomer loss for BiS4 and BiS5 at incubated at 40° C. for pH 7.5samples. BiS4 samples showed more rapid rate of loss of monomer comparedto BiS5 (FIG. 75A). The rate of monomer loss for BiS4 and BiS5 at pH 7.5was 27.4%/month and 4.5%/month, respectively (FIG. 75A). For BiS4,majority of monomer loss is due to fragmentation which was 23.9%/monthand to a lesser extent due to aggregation which was 3.5%/month (FIGS.75B and 75C). Interestingly, for BiS5, aggregation seemed to be atslightly higher rate (2.8%/month) compared to fragmentation rate(1.7%/month) (FIGS. 75B-75C).

Further analysis of the effect of pH on physical and chemical stabilityof BiS4 and BiS5 formats, the rate of monomer loss, fragmentation andaggregation per month was performed by plotting those values against the6 pH conditions (FIGS. 76A-76C). Throughout all six pH conditionsranging from pH 5.0-7.5, the rate of monomer loss was lower for the BiS5format compared to BiS4 (FIG. 76A), which suggests that the BiS5 formatdisclosed herein possesses unexpectedly superior physical and chemicalstability to other bispecific protein formats. For BiS4, the majority ofmonomer degradation was due to fragmentation even at lower pH conditions(FIG. 76B). BiS5 showed lower fragmentation rates compared to BiS4 inall the pH conditions tested. Surprisingly, the fragmentation rates inBiS5 seemed to be flat and lower across wider pH range compared to BiS4.Without being bound by any particular theory, it may be that the lowerfragmentation rates observed in BiS5 may arise from the G45 linkers oneither end of the scFv connecting it to the Fc. Fragmentation on one ofthe G45 linkers connecting to Fc, may not release the scFv, as it may bestill connected to the Fc via the other G45 linker. In BiS4 and BiS5,aggregation rates seemed to be similar throughout all the pH conditionstested (FIG. 76C), suggesting that the location of scFv has minimaleffect on aggregation kinetics, which is also supported by the observedno change in the T_(onset) between the two formats at all pH conditionsas measured using capillary DSC (FIG. 73E and Table 14, discussedabove). At pH 7.5 and 40° C. (at time=0), neither molecule exhibitedappreciable fragmentation (FIG. 77A), but under the same conditionsafter 2 weeks storage at 40° C., appreciable fragmentation is observedfor BiS4 and minimal fragmentation for BiS5 (FIG. 77B). Both BiS4 andBiS5 have reduced fragmentation and aggregation at lower (5.5) pH, whileBiS5 has superior performance at both pH values for both fragmentationand aggregation (FIG. 78). This series of experiments demonstrate thatthe BiS5, disclosed herein, possesses superior chemical stability andsimilar physical stability to that of BiS4.

Example 3.2

Additional studies were performed in order to evaluate the physical andchemical stability of different embodiments of the bispecific bindingproteins that are disclosed herein and identified as Constructs A-H(see, e.g., Table 13 and related Examples, above). These constructs wereanalyzed using DSC, accelerated storage stability, and FcRn and FcgRbinding assays, as follows below.

Differential Scanning Calorimetry Analysis

The DSC experiments for this data set were performed using a MicrocalVP-DSC scanning microcalorimeter (Microcal). All solutions and samplesused for DSC were filtered using a 0.22 μm filter and degassed prior toloading into the calorimeter. Antibodies used for the DSC studieswere >98% monomeric as determined by analytical SEC. Prior to DSCanalysis all samples were exhaustively dialyzed (at least 3 bufferexchanges) in 25 mM histidine-HCl (pH 6.0). Buffer from this dialysiswas used as reference buffer for subsequent DSC experiments. Prior tosample measurement, baseline measurements (buffer versus buffer) weresubtracted from the sample measurement. Dialyzed samples (at aconcentration of 1 mg/ml) were added to the sample well and DSCmeasurements were performed at a 1° C./min scan rate. Data analysis anddeconvolution were carried out using the Origin™ DSC software providedby Microcal. Deconvolution analysis was performed using a non-2-statemodel and best fits were obtained using 100 iteration cycles. TheT_(onset) is defined as the qualitative temperature at which thethermogram appears to have a nonzero slope, The T_(m) is defined as thetemperature at which half of the molecules in a set are unfolded, and iscalculated as the temperature value corresponding to each peak maximumon the thermogram.

The results for the different constructs are presented in FIG. 79.Generally, constructs A, C, D that include 2F4 as scFv have lower T_(M)1when compared to constructs E, G, H that include LC10 as scFv. Withoutbeing bound by theory, the difference in the TM1 value may be due to aninherently better thermal stability of the LC10 scFv domain relative tothe 2F4 variable domain. The data suggest that constructs A-D, having2F4 as scFv, would be less thermally stable than constructs E-H withLC10 as scFv.

Accelerated Storage Stability Analysis

The concentrations of the constructs were normalized to 1 mg/mL. 1 mL ofeach bispecific construct or IgG control was aliquoted into 1.5 mlEppendorf tubes. Samples were incubated in a static incubator for 2weeks at 45° C. Samples were analyzed at 3, 7, and 14 days and assessedfor stability. At each time point, a visual inspection was performed torecord any increased turbidity or precipitation. The samples werefiltered using a 0.2 um spin column and 120 ul of sample was aliquotedinto a HPLC vial, making sure there is no air bubble left at the bottomof the vial. Samples were then tested on an Agilent 1100 series HPLC-SECto check for aggregation and degradation using a TSK-GEL G3000SW_(XL)(300×7.8 mm) Tosoh Bioscience column with 0.1M sodium phosphate, 0.1Msodium sulphate, pH6.8 as the running buffer. 60 μL of sample wasinjected and run at a flow rate of 1 mL/min. The monomer retention time(mins), Total peak area, % monomer, % aggregate, % fragment, % monomerloss were captured and used for analytical SEC analysis. The results aresummarized in Table 15.

TABLE 15 Accelerated stability studies. T_(M)1 Monomer % Monomer %Aggreg % Aggreg % Degrad % Degrad % Construct (° C.) Day 0 Day 0 Day 0Day 7 Day 0 Day 7 A 57.11 97.1 68.4 2.2 30.0 .64 1.7 C 59.01 80.4 79.79.7 20.4 0.0 0.0 D 58.82 92.2 90.8 7.2 8.6 0.5 0.6 E 65.83 91.3 91.2 1.70.0 7.0 8.2 G 68.7 99.3 99.1 0.0 0.0 0.7 0.9 H 67.5 99.4 99.2 0.0 0.00.6 0.8

As described herein, the location of the scFv domain in the aboveconstructs is as follows (where the “-” indicates the scFv): A and E areat IS-RTP; B and F are at AK-GQP; C and G are at S-NG; and D and H areat SN-G. The various T_(M) values are associated with the followingdomains, T_(M)1=CH2/scFv; T_(M)2=Fab; T_(M)3=CH3. The data tend to showthat constructs A and C with 2F4 scFv inserted into ISRTP (A) and SNG(C) loops are more prone to aggregation than are constructs E and G thathave the LC10 scfv inserted at the same locations. This observationsuggests that the sequence identity and behavior of the scFv domain canhave an effect on the stability of the bispecific binding proteinconstructs. Further, from the above, it could be predicted thatconstruct D, which contains the 2F4 scFv, would have lower stabilitysimilar to A and C; however it seems that inserting the 2F4 scFv intothe SNG loop stabilizes the molecule and reduces tendency to formaggregates. Taken together this accelerated stability study indicatesthat scFv sequence and location within the Fc region can play ameasurable role in the stability of the BiSAb construct.

FcRn and FcγR Binding Analysis

Binding experiments were carried out using a BIAcore 3000 instrument(BIAcore). To capture the antibody, 1000 RU IsdH (Fab) antigen wasimmobilized on a CM5 chip. 100 nM of the BiSAb construct or mAb controlswere flowed at 20 μL/min for 5 min to capture antibody. 5 uM huFcRn orFcγR I, IIa, IIb, IIIa-158V or IIIA158F were flowed at 5 μL/min for 20min. FcRn binding was performed in PBS+5 uM EDTA at pH 6.0 while FcRnbinding was performed in PBS+5 uM EDTA at pH 7.4.

Constructs A, C, D, E, G and H were evaluated for FcRn binding.Representative data are shown in FIG. 80 for each of the bispecificconstructs E and H, and for 2F4 IgG binding to FcRn. The constructshaving scFv downstream of the CH2-CH3 interface appear to retain FcRnbinding (e.g., D & H constructs). Constructs having scFv located withinthe ISRTP loop upstream of the CH2-CH3 interface appear to removedetectable FcRn binding (e.g., A & E constructs). The ISRTP loop iswithin the region of known half-life extending YTE mutations in the Fc(M252Y/S254T/T256E) that are known to be important for FcRn binding.

Constructs A, C, D, E, G, and H were tested for binding to FcγRI,FcγRIIa, FcγRIIb, FcγRIIIa-158F, and FcγRIIIa-158V. Representative dataare shown in FIG. 81 for constructs E, G, and H binding toFcγRIIIa-158V. All constructs tested retained binding to FcγRs, thoughwith different affinities (FIG. 81, inset). Table 16 shows the observedbinding trends of the various constructs to FcγRs.

TABLE 16 FcγR binding trends. FcγRI FcγRIIa FcγRIIb FcγRIIIa158FFcγRIIIa158V D > C > D > C > A D > C > A D > C > A D > C > A A H > G >H > G > E H > G > E H > G > E H > G > E E

The differences observed in FcγR binding with constructs having scFvinserted in the ISRTP loop upstream of CH2-CH3 interface (A and E) showconsistently reduced FcγR binding when compared to the other constructshaving the scFv inserted into the SNG loop downstream of the CH2-CH3interface (C, D, G, and H).

Attempts to evaluate whether FcRn binding in the constructs A and Ecould be improved or restored were made by introducing a half-lifeextending loop (N3) to the Fc region. FIG. 82 is representative of thedata and shows that for the E constructs, neither BiS5Ab E nor constructE with the N3 loop introduced (BiS5Ab E+N3) were able to bind FcRn.Furthermore, inserting the LC10 scFv into the N3 loop (N3 scFv) andkeeping the ISRTP loop intact also diminished FcRn binding belowdetectable levels. These data indicate that at least for the Econstruct, if not for each construct disclosed herein, both the ISRTPloop and N3 loop, if present, need to be intact and unmodified in orderto retain FcRn binding.

Example 3.3

In addition to the comparison between BiS4 and the bispecific bindingconstructs disclosed herein (BiS5), a study was performed in order toevaluate three other BiS structural motif platforms, identified as BiS1,BiS2 and BiS3 (see, FIG. 83). As will be appreciated by reference toFIG. 83, these platforms vary in terms of the location of one of thebinding domains (illustrated as an scFv domain). Of the five motifs,only BiS4 and BiS5 include two linker moieties as points of attachmentto the larger protein, the others (BiS1, BiS2, and BiS3) are attached bya single linker.

Briefly, representative molecules of each construct were analyzed forstability using the techniques discussed above in Examples 3.1 and 3.2.Samples of each construct were added to buffers of pH 5.0, 5.5, 6.0,6.5, 7.0, and 7.5 and were stored at 40° C. over a period of two months.The samples were then analyzed for fragmentation rate (FIG. 84),aggregation rate (FIG. 85), and monomer loss rate (FIG. 86) usingHP-SEC. Under these conditions, the analysis indicated that thebispecific binding protein format disclosed herein (“BiS5”; and in D/Hformat as denoted above in Table 13) had superior physical and chemicalstability relative to all the other formats at all pH conditions.

The SEC data were also used to map the various peaks to thecorresponding fragments of the BiS molecules (FIG. 87). The mapping wasbased on assumptions that include that fragmentation occurs in the hingeand linker region of the molecules, the size of the fragment, thetheoretical fragmentation, and how the expected fragment species alignwith respect to the fragment species observed in other formats. Whilethere is good resolution between lower molecular weight fragments (LMWF)in each format, there is also poor or no resolution between monomer andhigher molecular weight fragments (HMWF) in all formats. Based on theinformation in Table 17, it was concluded that the HP-SEC techniqueunderestimates the fragmentation in BiS formats to a greater extent thanfor monoclonal antibodies. An alternate analysis was developed asdiscussed below.

TABLE 17 Fragmentation analysis - SEC underestimates fragmentation ratesof BiS formats. Mol. Wt. RT* % frag. per month at 40° C. (2 months)Species (kDa) (min) BiS1 BiS2 BiS3 BiS4 BiS5 BiSAb monomer¹ 200 7.9 3.43.4 0.8 6.2 0.5 2Fab + 1scFv + Fc² 175 7.9  x (k₂)  x (k₂)  x (k₂)1Fab + 2scFv + Fc² 150 7.9  x (k₁)  x (k₂′)  x (k₁) 1Fab + 1scFv + Fc³125 8.7 1.7 (k₁) 0.8 (k₁) — 3.4 (k₁′) — 1Fab + 1scFv 75 9.7 0.9 (k₁) 1.0(k₁) — 1.5 (k₁′) — 1Fab 50 10.6 — — 0.4 (k₁) 1.2 (k₂′) 0.5 (k₁) 1scFv 2511.0 0.8 (k₂) 0.6 (k₂) 0.4 (k₂) — — *Retention Time ¹Monomer lossincludes loss due to fragmentation only and does not includeaggregation. ²Rates may be underestimated due to co-elution of HMWF withmonomer. ³Rates for the shoulder peak may vary because of drop downintegration.

An alternative analysis was developed in order to calculatefragmentation rates of HMWF using a molar extinction coefficient basedon the assumptions that (i) during degradation, if a small fragment isdetected then there should also be a corresponding large fragmentpresent; (ii) secondary fragmentation (fragments of fragments) does notsignificantly occur during the duration of the stability study; and(iii) fragmentation occurs in the linker region and/or the hinge region.Fragmentation rates were determined based on the followingrelationships:

${Monomer} = \begin{matrix}{LMWF} & + & {HMWF} \\k_{t} & k_{LMWF} & k_{HMWF} \\{mEC}_{m} & {mEC}_{LMWF} & {mEC}_{HMWF}\end{matrix}$$k_{HMWF} = {\frac{k_{LMWF}}{{mEC}_{LMWF}} \times {mEC}_{HMWF}}$

Further an analysis of fragmentation rates was conducted with theconstructs under reducing conditions, in order to identify whetherformation of disulfide bonds had an effect on fragmentation andstability. Representative data of this assay are presented in (FIG. 86).Under reducing conditions, higher fragmentation rates were observablefor all BiS formats, except for BiS1 (Table 19). It was concluded thatthe higher fragmentation rates under reducing conditions confirms thatthe scFv moiety in the BiS5 construct is tethered to the CH3 region(FIG. 89).

TABLE 20 Overview of fragmentation analyses. % frag. per month at 40° C.(2 months) Analytical Method BiS1 BiS2 BiS3 BiS4 BiS5 Std. HPSEC 3.4 3.40.8 6.4 0.5 Alt. analysis of std. HPSEC 6.7 5.9 4.3 9.9 2.3 Non-red.GXII 3.0 2.7 2.0 6.4 1.2 Red. GXII 2.3 3.9 3.3 8.8 6.1 Total no. of G₄Slinker/molecule 4 4 4 6 6

The characteristics of the stabilizing disulfide bonds disclosed abovewere further investigated. Results are shown in Tables 23 and 24 below.Bispecific antibodies corresponding to two different specificities weregenerated in BiS4 and BiS5 (scFv inserted in SN-G loop) formats with andwithout the stabilizing disulfide bond in the scFv. Acceleratedstability study indicated that BiS4 constructs without a stabilizingdisulfide bond had substantial monomer loss due to degradation which wasprevented by introducing the stabilizing VL-VH disulfide bond. Theseresults indicate that removal of stabilizing disulfide bond in the scFvin the BiS5 construct did not have a significant effect on itsstability.

TABLE 21 Anti-EGFR IgG/ Monomer % Monomer % Aggregation % Aggregation %Degradation % Degradation % anti-Her2 scFv T_(M)1 (° C.) Day 0 Day 14Day 0 Day 14 Day 0 Day 14 BiS5 with DSB 71.17 100 100 0.0 0.0 0.0 0.0BiS5 without DSB 69.83 95.1 97.0 4.9 3.0 0.0 0.0 BiS4 with DSB 72.28 10098.4 0.0 0.0 0.0 1.6 BiS4 without DSB 69.34 95.7 86.7 4.3 2.3 0.0 11.1

TABLE 22 D-LC10 IgG/ Monomer % Monomer % Aggregation % Aggregation %Degradation % Degradation % 2F4 scFv T_(M)1 (° C.) Day 0 Day 14 Day 0Day 14 Day 0 Day 14 BIS5 with DSB 58.82 92.2 89.6 7.2 9.7 0.5 0.7 BiS5without DSB 54.22 92.4 86.1 4.4 10.1 3.0 3.9 BIS4 with DSB 66.74 99.098.2 1.0 0.0 0.0 1.9 BIS4 without DSB 56.10 97.7 83.9 2.0 7.7 0.3 8.5

Based on all the above data it seems that the bispecific binding proteinformat disclosed herein is the most stable of all the formats tested.Furthermore, the BiSAb5 appears to be most stable at the lower pH valuestested (e.g., 5.0, 5.5, and 6.0) in terms of minimizing bothfragmentation and aggregation. As such the unexpected and surprisingstability characteristics of the BisAbs disclosed herein provide afurther advantage relative to other structural platforms and formatsthat are used in engineering bispecific binding molecules.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific aspects of the subject disclosure have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the disclosure will become apparent to those skilled inthe art upon review of this specification and the claims below. The fullscope of the disclosure should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

What is claimed is:
 1. A protein, comprising: a first binding domain(BD1) that binds to a first epitope, a second binding domain (BD2) thatbinds to a second epitope, and an Fc region comprising C_(H)2 and C_(H)3domains; wherein the Fc region comprises BD2 at a solvent exposed loopin the C_(H)2 domain, the CH3 domain, or at the interface of the C_(H)2and C_(H)3 domains; and wherein the protein is bivalent for binding toeach of the first and second epitopes.
 2. The protein of claim 1,wherein the Fc region comprises BD2 at a solvent exposed loop in theamino acid sequence in the C_(H)2 domain, the C_(H)3 domain, or at theinterface of the C_(H)2 and C_(H)3 domain.
 3. The protein of claim 2,wherein the solvent exposed loop comprises an amino acid sequence fromthe C_(H)2 domain.
 4. The protein of claim 3, wherein the solventexposed loop comprises the amino acid sequence ISRTP (SEQ ID NO:39). 5.The protein of claim 2, wherein the solvent exposed loop comprises anamino acid sequence from the C_(H)3 domain.
 6. The protein of claim 5,wherein the solvent exposed loop comprises the amino acid sequence SNG.7. The protein of claim 2, wherein the solvent exposed loop comprises anamino acid sequence from the interface of the C_(H)2 domain and theC_(H)3 domain.
 8. The protein of claim 7, wherein the solvent exposedloop comprises the amino acid sequence AKGQP (SEQ ID NO:40).
 9. Theprotein of claim 1 wherein BD2 comprises a single-chain variablefragment (scFv).
 10. The protein of claim 1 wherein BD1 comprises abinding domain selected from the group consisting of a Fab domain, anscFv, a single domain antibody, and an antibody variable domain.
 11. Theprotein of any claim 1, wherein BD1 comprises a Fab domain.
 12. Theprotein of claim 11, wherein the Fab domain is connected to the Fcregion via an antibody hinge region.
 13. The protein of claim 1, whereinthe Fc region comprises a domain selected from the group consisting ofan Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. 14.The protein of claim 13, wherein the Fc region comprises a variant Fcregion.
 15. The protein of claim 13, wherein the Fc region isaglycosylated.
 16. The protein of claim 13, wherein the Fc region isdeglycosylated.
 17. The protein of claim 13, wherein the Fc region hasreduced fucosylation or is afucosylated.
 18. The protein of claim 1wherein the protein further comprises a protein linker L1 between BD2and the Fc region.
 19. The protein of claim 1 wherein the proteinfurther comprises a first protein linker, L1, and a second proteinlinker, L2, between BD2 and the Fc region.
 20. The protein of claim 1wherein BD2 is associated with the Fc region via a protein linker L1.21. The protein of claim 1 wherein BD2 is associated with the Fc regionvia two protein linkers, L1 and L2.
 22. The protein of claim 19, whereinL1 and L2 are independently selected from (G₄S)₂ (SEQ ID NO:41), (G₄S)₃,(SEQ ID NO:42), and (G₄S)₄ (SEQ ID NO:43).
 23. The protein of claim 1,wherein the protein comprises a chimeric heavy chain comprising thefollowing polypeptide domains, from N-terminus to C-terminusV_(H)1-C_(H)1-C_(H)2(N-term)-BD2-C_(H)2(C-term)-C_(H)3; and BD1comprises a Fab domain; wherein V_(H)1 comprises a heavy chain variabledomain of the Fab domain and C_(H)1 comprises the heavy chain constantdomain 1 of the Fab.
 24. The protein of claim 1, wherein the proteincomprises a chimeric heavy chain comprising the following polypeptidedomains, from N-terminus to C-terminus V_(H)1-C_(H)1-C_(H)2-BD2-C_(H)3;and BD1 comprises a Fab domain; wherein V_(H)1 comprises a heavy chainvariable domain of the Fab domain and C_(H)1 comprises the heavy chainconstant domain 1 of the Fab.
 25. The protein of claim 1, wherein theprotein comprises a chimeric heavy chain comprising the followingpolypeptide domains, from N-terminus to C-terminusV_(H)1-C_(H)1-C_(H)2-C_(H)3(N-term)-BD2-C_(H)3(C-term); and BD1comprises a Fab domain; wherein V_(H)1 comprises a heavy chain variabledomain of the Fab domain, C_(H)1 comprises the heavy chain constantdomain 1 of the Fab.
 26. The protein of claim 1, wherein BD2 comprisesan scFv.
 27. The protein of claim 26, wherein the scFv comprises, fromN-terminus to C-terminus, V_(H)2-polypeptide linker-V_(L)2 orV_(L)2-polypeptide linker-V_(H)2; wherein V_(H)2 comprises the heavychain variable domain of the scFv and V_(L)2 comprises the light chainvariable domain of the scFv.
 28. The protein of claim 1 wherein theprotein further comprises a protein linker L1 between BD2 and the Fcregion.
 29. The protein of claim 1 wherein the protein further comprisesa first protein linker, L1, and a second protein linker, L2, between BD2and the Fc region.
 30. The protein of claim 1, wherein BD2 is associatedvia a linker (L1) to the C_(H)2 domain, the C_(H)2 domain, or theinterface of the C_(H)2 and C_(H)3 domains of the Fc region.
 31. Theprotein of claim 1, wherein BD2 is associated via two protein linkers,L1 and L2 to the C_(H)2 domain, the C_(H)2 domain, or the interface ofthe C_(H)2 and C_(H)3 domains of the Fc region.
 32. The protein of claim29, wherein L1 and L2 are independently selected is from protein linkershaving a length of 1-25 amino acids.
 33. The protein of claim 29,wherein L1 and L2 are independently selected from (G₄S)₂ (SEQ ID NO:41),(G₄S)₃, (SEQ ID NO:42), and (G₄S)₄ (SEQ ID NO:43).
 34. The protein ofclaim 1, wherein the first and second epitopes are different.
 35. Theprotein of claim 1, wherein the first and second epitopes are the same.36. A bispecific binding protein that binds to PD-1 and CTLA-4comprising a first peptide comprising the amino acid sequence of SEQ IDNO:1, and a second peptide the amino acid sequence of SEQ ID NO:2.
 37. Abispecific binding protein that binds to PD-1 and CTLA-4 comprising afirst peptide comprising the amino acid sequence of SEQ ID NO:3, and asecond peptide comprising the amino acid sequence of SEQ ID NO:4.
 38. Abispecific binding protein that binds to PD-1 and CTLA-4 comprising, afirst peptide comprising the amino acid sequence of SEQ ID NO:5, and asecond peptide comprising the amino acid sequence of SEQ ID NO:6.
 39. Abispecific binding protein that binds to PD-1 and CTLA-4 comprising afirst heavy chain comprising the amino acid sequence of SEQ ID NO: 9, afirst light chain comprising the amino acid sequence of SEQ ID NO: 7, asecond heavy chain comprising the amino acid sequence of SEQ ID NO: 12,and a second light chain comprising the amino acid sequence of SEQ IDNO:
 4. 40. A bispecific binding protein that binds to PD-L1 and CTLA-4comprising a first peptide comprising the amino acid sequence of SEQ IDNO:14 and a second peptide comprising the amino acid sequence of SEQ IDNO:15.
 41. A bispecific binding protein that binds to PD-L1 and CTLA-4comprising a first peptide comprising the amino acid sequence of SEQ IDNO:16, and a second peptide comprising the amino acid sequence of SEQ IDNO:17.
 42. A bispecific binding protein that binds to PD-L1 and CTLA-4comprising a first peptide comprising the amino acid sequence of SEQ IDNO:18, and a second peptide comprising the amino acid sequence of SEQ IDNO:19. 43-51. (canceled)
 52. A composition comprising the protein ofclaim 1 and a pharmaceutically acceptable carrier.
 53. A nucleic acidmolecule comprising a nucleotide sequence encoding a protein of claim 1.54. A vector comprising the nucleic acid molecule of claim
 53. 55. Ahost cell comprising the vector of claim
 54. 56. A method of treating orpreventing cancer in a subject, the method comprising administering theprotein of claim 1 to the subject.
 57. The method of claim 56, whereinthe cancer is one or more of ovarian cancer, breast cancer, colorectalcancer, prostate cancer, cervical cancer, uterine cancer, testicularcancer, bladder cancer, head and neck cancer, melanoma, pancreaticcancer, renal cell carcinoma, and lung cancer.
 58. A method of enhancingan immune response in a subject, the method comprising administering theprotein of claim 1 to the subject.